Diagnostic and therapeutic targeting of pathological tau proteins in neurodegenerative disorders

Tauopathies, characterized by fibrillar tau accumulation in neurons and glial cells, constitute a major neuropathological category of neurodegenerative diseases. Neurofibrillary tau lesions are strongly associated with cognitive deficits in these diseases, but the causal mechanisms underlying tau‐induced neuronal dysfunction remain unresolved. Recent advances in cryo‐electron microscopy examination have revealed various core structures of tau filaments from different tauopathy patients, which can be used to classify tauopathies. In vivo visualization of tau pathology is now available using several tau positron emission tomography tracers. Among these radioprobes, PM‐PBB3 allows high‐contrast imaging of tau deposits in the brains of patients with diverse disorders and tauopathy mouse models. Selective degradation of pathological tau species by the ubiquitin‐proteasome system or autophagy machinery is a potential therapeutic strategy. Alternatively, the non‐cell‐autonomous clearance of pathological tau species through neuron–glia networks could be reinforced as a disease‐modifying treatment. In addition, the development of neuroinflammatory biomarkers is required for understanding the contribution of immunocompetent cells in the brain to preventing neurodegeneration. This review provides an overview of the current research and development of diagnostic and therapeutic agents targeting divergent tau pathologies.

Tauopathies, characterized by fibrillar tau accumulation in neurons and glial cells, constitute a major neuropathological category of neurodegenerative diseases.Neurofibrillary tau lesions are strongly associated with cognitive deficits in these diseases, but the causal mechanisms underlying tau-induced neuronal dysfunction remain unresolved.Recent advances in cryo-electron microscopy examination have revealed various core structures of tau filaments from different tauopathy patients, which can be used to classify tauopathies.In vivo visualization of tau pathology is now available using several tau positron emission tomography tracers.Among these radioprobes, PM-PBB3 allows high-contrast imaging of tau deposits in the brains of patients with diverse disorders and tauopathy mouse models.Selective degradation of pathological tau species by the ubiquitinproteasome system or autophagy machinery is a potential therapeutic strategy.Alternatively, the non-cell-autonomous clearance of pathological tau species through neuron-glia networks could be reinforced as a diseasemodifying treatment.In addition, the development of neuroinflammatory biomarkers is required for understanding the contribution of immunocompetent cells in the brain to preventing neurodegeneration.This review provides an overview of the current research and development of diagnostic and therapeutic agents targeting divergent tau pathologies.
Accumulation of intracellular neurofibrillary tangles (NFTs) consisting of microtubule-associated protein tau is a major hallmark of Alzheimer's disease (AD) and related neurogenerative diseases, collectively referred to as tauopathies [1][2][3].In AD, tau depositions are noted in neurons as somatodendritic NFTs, neuropil threads, and dystrophic neurites encompassing senile plaques, and the loss of neurons in specific regions coincides with the progression of NFTs [4].The distribution of NFTs throughout the brain is correlated with the severity of cognitive impairment [5].Hence, NFTs and/or tau assembly at an earlier fibrillogenesis stage are considered to be toxic species.Although the identity of the exact neurotoxic tau species remains unclear, studies of experimental models suggest that NFTs themselves may not be neurotoxic [6][7][8][9].NFT formation is initiated by the conversion of natively unfolded tau protein into insoluble tau aggregates through dimerization, oligomerization, and protofibril formation and is tightly associated with neurodegenerative processes.It is possible that a specific structural change of tau molecules from a physiological to a disease state provokes neurotoxicity at an early stage of neurodegeneration.The identification of cryo-electron microscopy (cryo-EM) structures of tau filaments from tauopathy brains may help to prove this hypothesis [10].
Nevertheless, it is essential to identify such a specific structure for therapeutic mitigation of neurotoxic insults.On the other hand, similar to other cerebral proteinopathies that are characterized by the existence of aggregated forms of proteins or peptides (e.g., amyloid-b, tau, a-synuclein, and TDP-43), dissecting aggregation processes may provide a universal strategy for the development of disease-modifying treatments against AD.Unlike other amyloid-forming proteins, tau protein does not undergo fibrillization in vitro without any inducer molecules (e.g., heparin, heparan sulfate, polyunsaturated fatty acid, RNA, or quinones).At present, the endogenous, physiological inducers of tau aggregation are not known yet and need to be discovered for unraveling the initiation of NFT formation.
Neurotoxic tau species may provoke neuronal cell death in a cell-autonomous manner.In general, intracellular protein degradation is important for the maintenance of protein metabolism and for preventing the accumulation of misfolded proteins [11].Tau protein metabolism is typically maintained by ubiquitinproteasome system (UPS) and autophagy-lysosome systems [12,13].Reducing pathological tau accumulation through these protein degradation systems could be one of the therapeutic strategies, although it remains elusive whether these systems can selectively process pathological but not physiologically functioning tau species.
Microglia are the resident phagocytes of the central nervous system (CNS), and their activation is considered to play an important role in the pathogenesis of neurodegenerative diseases.Recent studies with singlecell RNA sequencing analysis of CNS cells in AD and other neurodegenerative conditions revealed that the transition from homeostatic microglia to diseaseassociated microglia (DAM) was defined by changes in the expression of characteristic genes [14,15].However, it is yet to be clarified whether and when changes in gene expression occur in response to the pathogenesis.Non-cell-autonomous mechanisms governed by DAM should be taken into account for preventing neurotoxicity injuries and death of pathological tau-bearing neurons.
The current research conducted on cryo-EM has revealed distinctive structural variations in the tau fibril core in diverse tauopathies.Furthermore, tau positron emission tomography (PET) imaging has demonstrated its potential in diagnosing tauopathies by analyzing the pathological distribution of tau deposits in living brains.In this review, we overview the recent research progress to clarify diverse tau assemblies and their implications for the diagnosis of various tauopathies.Additionally, we present insights into the degradation systems acting on pathological tau species from the perspective of protein homeostasis.Finally, we discuss the potential link between tau-induced neurodegeneration and microglial functions, as microglia play a crucial role in maintaining brain homeostasis and promoting neuronal deteriorations and may be a key target for disease-modifying therapies.

The tau protein
The tau protein belongs to the family of Tau/MAP2/ MAP4 microtubule-associated proteins.In the CNS, tau is mostly expressed in neurons but is also present at low levels in glia [16].Alternative splicing of MAPT in the adult human brain generates six tau isoforms [17] (Fig. 1A).These isoforms range from 352 to 441 amino acids in length and differ by the presence or absence of inserts of 29 or 58 amino acids (encoded by exons 2 and 3) in the N-terminal half, and the inclusion or exclusion of the 31 amino acid microtubulebinding repeat (encoded by exon 10) in the C-terminal half [18] (Fig. 1A).The inclusion of exon 10 results in the production of three tau isoforms with four microtubule-binding repeats (4R) and its exclusion in other three isoforms with three repeats (3R).In the adult human brain, similar levels of 3R and 4R tau isoforms are expressed [19].Tauopathies can be classified into the following three groups on the basis of the tau isoforms found in the aggregates from diseased brains: (1) 4R tauopathies, including progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and argyrophilic grain disease (AGD); (2) 3R tauopathies, including Pick disease (PiD); and (3) 3R + 4R tauopathies, including AD and chronic traumatic encephalopathy (CTE) [20,21] (Fig. 1A-C).Dominantly inherited mutations in MAPT cause a form of frontotemporal dementia that can be associated with parkinsonism (FTDP-17T).To date, more than 80 mutations have been identified in either exonic or intronic regions of human MAPT (Alzforum website) and can be classified into missense and splicing mutations.The majority of missense mutations cluster around the microtubule-binding domain.Most splicing mutations are within or near intron 10, increasing the inclusion of exon 10 and consequently the ratio of 4R tau to 3R tau, although there are several exceptions such as DK280, L266V, and G272V mutations that increase 3R versus 4R tau isoforms.In human brains, the imbalance between the 3R and 4R tau may be a key event to cause tauopathies, but the mechanisms underlying this alteration are still unknown.Importantly, tau isoform expression is not conserved between species [22,23].Adult mice express predominantly 0N4R isoform, while rats have 0N4R, 1N4R, and 2N4R with the same ratios [24].In mice, 3R isoform is mainly expressed during the embryonic stage and is replaced by 4R tau between postnatal days 9 and 18 [25].This isoform switching most likely induces axonal elongation and neuronal development.
To search physiological functions of tau, several lines of tau-knockout mice were extensively examined [26].Early investigations showed that tau-knockout mice presented no overt abnormal phenotypes [27][28][29].However, recent studies have revealed several pathological changes with behavioral abnormality in tau-knockout mice [30][31][32].In contrast to the indispensability of tau, deficiency of tau protected against Ab-induced excitotoxicity [33,34], indicating the involvement of endogenous tau in regulating neuronal activity under pathological circumstances.However, it is still unknown whether tau plays a significant role in physiological network activity because these experiments were performed under excessive expressions of the human amyloid precursor protein.In addition, research on tau-knockout mice has shown that tau may be involved in neurogenesis [35], iron export [31], and long-term depression (LTD) [36].

Neuropathological features of tau inclusions
Although tauopathies share a common molecular mechanism, histopathological features vary across disease types [37].Pathological features of AD brains are NFTs composed of paired helical filaments (PHFs) and straight filaments (SFs).Immunostaining of AD brain slices with antibodies against phosphorylated tau (p-tau) labels NFTs, neuropil threads, and dystrophic neurites surrounding Ab deposits (i.e. neuritic plaques).PSP, CBD, and PiD are non-AD tauopathies with focal cortical and/or subcortical neuronal loss and gliosis.These non-AD tauopathies are categorized in the spectrum of sporadic frontotemporal lobar degeneration with tau pathology (FTLD-tau).Pathological features of PSP brains are globose NFTs, tufted astrocytes, and oligodendrocytic coiled bodies observed in the subthalamic nucleus, globus pallidus, ventral thalamus, cerebellar dentate nucleus, and cerebral cortex [38].Ultrastructural assessments showed that tau assemblies in PSP were mostly composed of SFs with rare twisted filaments (Fig. 1B).CBD is one of the 4R tauopathies with neuronal and glial hyperphosphorylated tau aggregates in both gray and white matter of the neocortex, basal ganglia, thalamus, and the brainstem [39].Astrocytic plaque is a unique glial pathology in CBD.Ultrastructures of tau aggregates in CBD are mostly SFs with some wide twisted filaments.Either PSP and CBD can be found as a neuropathological background in diverse FTLD syndromes, including corticobasal syndrome, PSP syndrome, frontotemporal dementia, and nonfluent/agrammatic primary progressive aphasia [40][41][42].Pick bodies forming round intraneuronal inclusions are the histopathological hallmark of PiD [43,44].These inclusions are composed of hyperphosphorylated 3R tau and are typically observed in hippocampal pyramidal neurons and granular neurons of the dentate gyrus.There are ballooned neurons and variable tau-immunoreactive glial inclusions [45].Ultrastructures of tau aggregates in PiD are mostly SFs with some wide twisted filaments (Fig. 1B).In AGD brains, grains are typically found in the neuropil of limbic areas and diffusely deposited in the cortex.Spindle-shaped 4R tau lesions in neuronal processes, coiled bodies in oligodendrocytes, and pretangles in neurons are also present in AGD brains [37,46].Neuropathological features of CTE are defined by the irregular cortical distribution of p-tau immunoreactive NFTs and astrocytic tangles with a prediction for the depth of cerebral sulci.3R + 4R tauopathy was confirmed by immunohistochemistry [47] and biochemistry [48].Ultrastructures of CTE tau filaments are predominantly helical filament type with projected widths of 20-25 nm and crossover spacings of 65-80 nm [48].These filaments differ from the PHFs and SFs of AD.
Current advances of cryo-EM examination revealed that tauopathies could be classified according to the core structures of tau filaments [10].Even if components of tau aggregates are similar between AD and CTE, the CTE tau filament fold is distinct from that of the AD tau filament (Fig. 1C).Based on cryo-EM observations, the 4R tauopathies are divided into two classes [10].The PSP tau filament fold comprises three-layered core regions, whereas filamentous structures from CBD and AGD brains are four-layered folds (Fig. 1C).The differences in the filament structures between these 4R tauopathies are consistent with the profiles of N-terminally truncated tau fragments examined by western blots of sarkosyl-insoluble tau [10].
Because tauopathies induce a broad range of symptoms such as behavioral, movement, language, and memory impairments [49,50], the clinical diagnosis and differentiations of these illnesses may not necessarily be in agreement with neuropathological classifications.The definitive diagnosis of tauopathies has been only enabled by examining the shapes and distribution of tau deposits, affected cell types, and tau isoform composition in autopsied brain samples.Now, cryo-EM assays can identify disease-characteristic filamentous structures of tau inclusions from postmortem brains, despite the lack of histological information due to the extractions of buffer-insoluble materials from tissues with a large volume.Such ultrastructural properties can also be associated with subcellular, cellular, and regional localizations and morphology of inclusions.

Neuroimaging-based diagnostic assessments of tauopathies
It is known that high levels of p-tau181 (tau phosphorylated at Thr181) and total tau have consistently been found in cerebrospinal fluid (CSF) of AD patients relative to healthy elderly controls [51].Since CSF p-tau181 levels in AD are higher than those in non-AD tauopathies, CSF p-tau181 is also a better indicator for differential diagnosis [52].Recent reports showed that relative levels of p-tau217 (tau phosphorylated at Thr217) in CSF were correlated with burdens of PETdetectable Ab and tau aggregates and CSF measures of Ab [53,54].Janelidze et al. also observed that CSF p-tau217 correlates stronger than CSF p-tau181 with PET measures of tau and amyloid pathologies in AD and hypothesized that p-tau217 levels may reflect the pathological state of tau better than p-tau181 levels, although the sensitivity of p-tau is highly dependent on the performance of antibodies (e.g., anti-p-tau217 antibody IBA413 and anti-p-tau181 antibody AT270) [54].More recently, it was reported that plasma p-tau (both p-tau181 and p-tau217) was able to discriminate AD from healthy control and FTLD [55][56][57].Compared with CSF analyses, blood-based biomarkers can be widely used in primary clinical settings as less invasive and equally cost-effective tools.
Imaging biomarkers are now available for detecting in vivo tau pathology with a panel of PET tracers.Tau tracers are designed according to b-sheet binding properties and labeled AD-type tau deposits, while these compounds exhibit differential reactivities with non-AD tau aggregates.By now, [ 11 C]PBB3, [ 18 F]PM-PBB3, [ 18 F]AV1451, [ 18 F]THK5351 (and its analogs), [ 18 F]MK-6240, [ 18 F]R06958948, [ 18 F]GTP-1, and [ 18 F] PI-2620 have been applied to human subjects [58][59][60][61][62][63][64][65] (Table 1).The distribution of the bound tracers recapitulated Braak NFT staging in AD [66].Firstgeneration tracers exemplified by [ 18 F]AV1451 and [ 18 F]THK5351 exhibit off-target effects such as binding to monoamine oxidase (MAO)-A and MAO-B [67,68], whereas second-generation tracers such as [ 18 F]PM-PBB3, [ 18 F]MK-6240, [ 18 F]R06958948, [ 18 F]GTP-1, and [ 18 F]PI-2620 seem to have less off-target binding [61,69,70].[ 11 C]PBB3 was designed to capture tau deposits in a wide range of tauopathies [58].This ligand reacts with 3R and 4R tau pathologies in human brains better than [ 18 F]AV1451 [71].[ 18 F]PM-PBB3, which was generated by modifying the chemical structure of PBB3 for relatively high metabolic stability, has the advantage of an 18 F-labeled probe over 11 C-radiotracers for broader availability and higher PET scan throughput.At present, [ 18 F]PM-PBB3 is the unrivaled tracer to capture diverse tau fibrils with different isoform compositions and conformations with contrast and dynamic range adequate for individual-based assessments of AD-and FTLDspectrum syndromes [65].Although the feasibility of fluid biomarker tau PET imaging for evaluating the severity of tau pathologies in AD patients still needs to be established by examining their correlation with neuropathological findings in postmortem assessments, the relationship between these two bioassay modalities has been indicated.Indeed, a mass-spectrometry-based assay recently demonstrated that the microtubulebinding region of tau containing the residue 243 (MTBR-tau243), p-tau205 and p-tau217 in CSF were associated with [ 18 F]AV1451-PET imaging and cognitive deficits [72].
The binding properties of tau PET tracers with tau fibrils are examined by computational modeling using structural information from cryo-EM studies [73,74] (Fig. 2A-C).The modeling identified several potential high-affinity binding sites with some diversities of binding reactivity for each tracer [73,74].To clarify whether different tau filamentous structures can be distinguished by tau PET tracers, Mishra et al. performed molecular modeling study on tau PET tracer binding to the core structure of PiD tau filament [75] (Fig. 2B-D).The examined tracers, including AV-1451, MK-6240, PBB3, PM-PBB3, THK5351, and PiB, bind to PiD tau filament fold at multiple surface binding sites and in a cavity binding site.Docking and molecular dynamics simulations revealed a unique binding site (the groove between R349 and Q351) for PBB3 and PM-PBB3 (Fig. 2C,D).To further confirm PM-PBB3 binding on the tau filament fold, a cryo-EM examination of the AD tau filament fold with PM-PBB3 was performed [76] (Fig. 2E,F).One of the two major binding sites was a groove between R349 and Q351, which commonly existed in PHFs and SFs.The groove between the side chains of Q351 and K353 contains two binding sites, and there are side-to-side interactions between PM-PBB3 compounds in parallel with the long helical axis of tau filaments (Fig. 2F).Interestingly, an additional binding site in a direction nearly perpendicular to the helical axis in SFs was observed though resolutions did not reach to define the orientation of PM-PBB3 (Fig. 2F).Due to less space in the cavity, perpendicular bindings of PM-PBB3 were not present in PHFs.Assumedly, perpendicular bindings of PM-PBB3 forming a high-density ladder amplify PET signals.Thus, tau fibrils in non-AD tauopathies such as PSP, CBD, CTE, and PiD could have similar properties of PM-PBB3 binding.It will be essential to identify the binding sites of PM-PBB3 in tau filaments from these non-AD tauopathies by cryo-EM examination in the future.Nevertheless, cryo-EM is a promising research tool for developing new PET tracers with higher specificity and affinity towards the highprecision differential diagnosis of tauopathies.

Targeting tau protein homeostasis for therapies
In general, the endoplasmic reticulum system, autophagylysosome system, and UPS are the three main regulatory pathways for maintaining protein homeostasis and preventing excess dysfunctional protein species.Several research groups have focused on the identification of pathways involved in the degradation of misfolded tau proteins, since such molecular machineries are likely to be impaired in the pathogenesis of tauopathies and are of critical significance as targets for disease-modifying therapies.Duff and her colleagues reported the selective vulnerability of excitatory neurons to tau pathology and observed higher levels of BCL2-associated athanogene 3 (BAG3), a facilitator of autophagy, in inhibitory neurons than in excitatory neurons [77].Insufficient clearance of excessive tau protein may cause excitatory neuronal cell death.These excessive tau species may form toxic conformers.As mentioned above, NFTs may not be the primary toxic tau species in the brains of patients with AD and other tauopathies [6], and researchers hypothesized that tau oligomers are responsible for a large part of disease-related neurotoxicity [78,79].When several tau species were injected into the mouse hippocampus, tau oligomers caused memory deficits and cell damage, while neither tau monomers nor tau fibrils caused any abnormality [80].Recently, we reported that the genetic ablation of p62/SQSTM1, a ubiquitinated cargo receptor for selective autophagy, exacerbates tau pathologies, neuronal death, and neuroinflammation in a mouse model of tauopathy (PS19 mice) [81].Immunolabeling analyses with an antibody selectively recognizing tau oligomers [82] demonstrated that PS19/p62-KO mice displayed accumulation of tau oligomers at a significantly high level than PS19 mice.Since p62 is the most abundant and major autophagy receptor in mouse brains [81], p62 likely exerts neuroprotection against tau pathologies by eliminating neurotoxic tau species (Fig. 3A).
The proteolysis-targeting chimeras (PROTACs), which bind to target misfolded proteins and E3 ubiquitin ligase to induce Lys48 polyubiquitination and proteasomal degradation of the target protein, is one of the novel technologies for eliminating neurotoxic components [83] (Fig. 3B).Chu et al. showed that taudegrading PROTAC generated by fusing a tau-binding motif from b-tubulin to E3 ligase-binding peptides promote tau degradation in an AD mouse model, 3xTg-AD [84].Another study documented a small-molecule PRO-TAC composed of tau PET tracer, AV-1451, and thalidomide, a ligand for Cereblon (CRBN, a substratereceptor for the E3-ubiquitin ligase CRL4 CREN ) [85].The concept of this study was to target diseaseassociated tau and to reduce the stress vulnerability of FTD neurons.In addition to PROTAC, autophagytargeting chimeras (AUTACs) to degrade target proteins through autophagy machinery have been developed.Takahashi et al. reported that AUTACs contained guanine-derived p-fluorobenzylguanine tag and Halo Tag (HT) ligand selectively degraded target proteins through Lys63-linked ubiquitination [86].Taken together, PROTAC or AUTAC is a heterobifunctional peptide or small molecule that simultaneously binds to target proteins and UPS components or autophagy machinery to remove those proteins or dysfunctional organelles such as mitochondria [83,86].Targeting ubiquitin signaling could be a fundamental strategy for ameliorating neuronal function and survival.

Targeting neuron-glia interactions for therapies
Microglia are the resident phagocytic cells in the CNS and play a critical role in pathological and physiological processes.Microglia express a wide range of receptors that act as molecular sensors recognizing intracellular and extracellular insults followed by immune responses.Activated microglia become highly motile, secreting inflammatory cytokines and phagocytosing cell debris or damaged neurons [87].To investigate the link between microglial activation and tauopathy, the tauopathy mouse model PS19 crossbred with TREM2 knockout mice was examined [88].The lack of TREM2 rescued brain atrophy and decreased expressions of DAM markers such as APOE and Cst7, implying the critical involvement of DAM in tau-induced neurodegeneration.It is also noteworthy that PET imaging of tauopathy mouse models, PS19 and rTg4510, showed an age-dependent increase of the mitochondrial 18-kDa translocator protein (TSPO), a marker of microglial activation, along with pathological tau accumulation and brain atrophy [89,90].These data raise a possibility that the upregulation of TSPO is associated with the deleterious roles of DAMs in these models.To investigate the potential of TSPO as a therapeutic target, the effects of treatment with a TSPO ligand, Ro5-4864, were examined on the rTg4510 mice [91].Ro5-4864 treatment attenuated brain atrophy, hippocampal neuronal loss, and levels of C1q, a regulator of the complement cascade [91] (Fig. 3A).In another study, the elimination of a complement, C3, in PS19 mice was performed to investigate possible linkage between complement pathway and tau-induced neurodegeneration [92].C3 knockout in the PS19 mice ameliorated neuron loss and brain atrophy and improved neurophysiological and behavioral measures [92].As Hansen et al. reported in their review article, reducing complement activation could be a potential therapeutic approach to tauopathies [93].It is still unclear whether microglial phagocytosis plays a beneficial or detrimental role in neurodegenerative diseases.However, effective clearance of neurotoxic components could be a potential therapeutic strategy for preventing neurodegenerative diseases.There are a number of receptor systems contributing to phagocytosis in the CNS [94].The TAM receptor tyrosine kinases, consisting of Tyro3, Axl, and Mer, have a critical function in macrophages and immune sentinels for the phagocytosis of apoptotic cells [95][96][97].TAM receptors recognize phosphatidylserine exposed on the cell surface, an eat-me signal, via protein S or GAS6 [98].A recent study revealed that TAM receptors, Axl and Mer, were required for microglial recognition and phagocytosis of Ab plaques [99].Microglial gene expression analysis in neurodegenerative disease mouse models, including App NL-G-F/NL-G-F , rTg4510, and SOD G93A mice, showed upregulation of Axl, but no change of Mer in all three mouse models [100].Hence, Axl could become a universal receptor for maintaining brain homeostasis by phagocytosis.Further characterization of TAM receptor-associated eat-me signaling in tauopathy will be necessary.
As a counterpart of DAM, the abundance of homeostatic microglia reflects the physiological, nondiseased status.Transcriptome analysis of microglia isolated from neurodegenerative disease models showed a reduction of homeostatic genes P2RY12, Tmem119, and CX3CR1.[14,100] In our hand, the rTg4510 mice showed the regression of P2RY12 protein level before the massive accumulation of intraneuronal tau deposits and an elevation of TSPO immunoreactivity [101].Since P2RY12 declines preceded increases of Iba1 and TSPO in the rTg4510 mice, this homeostatic marker would be a sensitive marker heralding the activation of deleterious microglia.A recent study showed neuroprotective functions of microglia through somatic neuron-microglia interaction by P2RY12 clustering in the microglial process [102].The cellular interaction was regulated by the purinergic signaling from neuronal mitochondria.Modulation of neuronal mitochondrial activity and/or purinergic signaling could lead to the maintenance of microglial homeostasis, although further studies should be performed to understand the significance of neuron-microglia interactions.

Neuroimaging-based drug development for preventing tauopathy
The drug development process should include target identification, drug screening, non-clinical tests in animal models, and clinical trials [103].The establishment of evaluation tools is of vital significance for adequate assessments of drug efficacies.In AD drug development, several biomarkers, such as brain imaging and CSF measures, can assist in diagnosis, demonstrate target engagement, and support disease modification.Of particular interest are imaging biomarkers, including amyloid PET and tau PET, as mentioned above.Tau PET tracers, [ 11 C]PBB3 and [ 18 F]PM-PBB3, are able to capture wide-range tau pathologies in AD, non-AD tauopathies, and model mice exemplified by rTg4510 and PS19 [58,65,90,104].Importantly, in vivo brain imaging enables longitudinal examinations of neurodegenerative processes and pathological tau formation [105].The use of animal models that recapitulate the critical features of the disease, such as NFTs, cognitive impairment, brain atrophy, and neuronal loss, greatly facilitates the evaluations of tau-targeting therapies.Recently, a couple of neuroimaging-based studies successfully demonstrated the efficacy of a TSPO ligand, Ro5-4864, and a low-protein diet on the rTg4510 mice [91,106].These studies showed suppression of brain atrophy measured by volumetric magnetic resonance imaging but no significant change of tau PET signals, suggesting that the neuroprotective effects of these treatments were on the downstream of tau accumulation.However, due to lower spatial resolution and partial volume effects in animal PET imaging, caution is warranted in interpreting findings in PET imaging of tauopathy mouse models.Moreover, radiosignals arising from off-target tracer binding [107] need to be considered in the analysis of non-clinical tau PET data.
There are limited numbers of animal models that display intracellular filamentous tau aggregations [108].As far as we know, several lines of P301L/S mutant tau-expressing transgenic mice developed neurofibrillary pathology in the CNS, whereas most non-mutant tau-expressing transgenic mice rarely developed tau pathology.Recent studies suggest that there is a nontau factor to induce neurodegeneration in the rTg4510 mice expressing P301L human tau due to transgene integrations into the coding sequence of mouse endogenous genes [109,110].Therefore, candidate drug interventions with current mouse models of tauopathy need to pay close attention to whether effects are really associated with tau-induced neurodegeneration.
Neuroinflammation is an inflammatory response in the CNS.The chronic over-activation of proinflammatory response has been implicated in many neurodegenerative diseases [111].The development of neuroinflammatory biomarkers is necessary to understand the contribution of such responses to the initiation and progression of tauopathies.In vivo visualization of activated microglia in humans and mice is available with the use of PET tracers of TSPO [90,112,113].In vivo imaging study of rTg4510 mice revealed that the increase of TSPO signal was a late event following pathological tau accumulation [90,114].To seek an early phenotypic change of microglia, other biomarkers will be needed.Currently, the DAM signature defined by RNA-seq analysis in 5xFAD mice [14] was generally accepted for staging the neurodegenerative phenotype of microglia.Once the DAM signature in the rTg4510 mice is discriminated, microglia stages can be linked to pathological stages defined by tau PET imaging (Fig. 4).
Candidates of neuroinflammatory biomarkers can be selected from microglia stage-dependent gene expressions (Fig. 4).Neuroinflammatory biomarkers identified in mouse models can be translated to humans, otherwise reverse translational research can be conducted to re-screen biomarkers.Eventually, these biomarkers will become powerful tools for drug development based on neuron-glia interactions.

Conclusion
To achieve disease-modifying therapies for tauopathies, it is crucial to identify an appropriate target to The DAM signature in tauopathy mouse models is examined for staging the tau-related neurodegenerative phenotype of microglia, and candidates of neuroinflammatory biomarkers can be selected from stage-dependent gene expressions.(3) Microglia pathological stages can be linked to tau pathology stages defined by tau PET imaging for further selection of tauopathy-related DAM markers, and this approach can be supplemented by immunohistochemical and biochemical assays of excised tissues.(4) Selective small-molecule, brain-entering PET radioligands for the selected molecular targets are developed by in-vivo imaging and in-vitro autoradiographic evaluations.PET images of tau pathologies and neuroinflammatory changes in this figure tentatively illustrate the performance of 18 F-PM-PBB3 and translocator protein (TSPO) tracer, 11 C-Ac5216, respectively, in rTg4510 mouse brains putatively at three different stages.However, new neuroinflammatory PET ligands will need to be generated as TSPO is not included in DAM makers.
modulate the disease progression.The formation of local NFTs is strongly linked to cognitive impairment, and as tau protein is a main constitute of NFTs, it should be a primary target for the development of drugs and diagnostics.Recent advancements in cryo-EM have enabled the identification of core structures of tau filaments in tauopathy brains, which can be used to classify different types of tauopathies.Tau PET imaging also holds the potential for diagnosing tauopathies by evaluating the distribution of pathological tau deposits in living brains.Additionally, interventions in protein degradation systems such as the UPS and autophagylysosome system, as well as microglial phagocytosis, are currently being investigated for their effectiveness as disease-modifying therapies.

Fig. 1 .
Fig. 1.Disease-associated characteristics of tauopathies.(A) Tauopathies can be classified into three groups: 3R + 4R tauopathies, including AD, 3R tauopathies, including PiD and 4R tauopathies, including PSP and CBD.(B) Representative electron microscopical images of tau assemblies in AD, PSP, and PiD brains.Inboxes show PHFs and SFs in AD, SFs and twisted filaments in PSP, and SFs with some wide twisted filaments in PiD.(C) The core structures of tau filaments observed by cryo-EM examination.AD and CTE are classified into 3R + 4R type [10].PiD is classified into 3R type.CBD, AGD, and PSP are classified into 4R type.The 4R types are divided into two classes.The PSP tau filament fold comprises three-layered core regions, whereas filamentous structures from CBD and AGD brains are four-layered folds.

Fig. 2 .
Fig. 2. Binding sites of tau PET probes in tau filament core.(A) Molecular docking of PBB3 into the PHF protofilament core structure [74].S1 site has the highest affinity for the tau filaments, followed by S2 and S3.(B) Binding sites of tau PET tracers in Pick filament core predicted by molecular docking (MD) [115].Secondary structures of Pick filaments (K254 to F378) show binding sites S1 to S11 (green) and cavity site C1 (blue).(C, D) Molecular interactions of PBB3 (C) and PM-PBB3 (D) with Pick filaments taken after 100-ns MD simulation [115].PBB3 binds to R349 and K347 sidechain hydrogens.PM-PBB3 binds to R349 and Q351 sidechain hydrogens.(E) AD core with PM-PBB3 binding sites [76].Major binding sites 1, 2a, 2b and 3 are shown in orange.Minor binding sites 4, 5, 6a and 6b are indicated in yellow.(F) Binding of PM-PBB3 to SFs [76].Top views and side views of extra densities in the PM-PBB3 binding sites.

Fig. 3 .
Fig. 3. Therapeutic targets for preventing tauopathies.(A) Process of pathological tau formation and neuronal death.The autophagy receptor p62 may trigger tau degradation by eliminating neurotoxic tau species.TSPO ligand Ro5-4864 may attenuate both microglia activation and neurodegeneration.(B) The PROTACs is one of the novel technologies for eliminating neurotoxic components.A degrader binds to target misfolded proteins and E3 ubiquitin ligase to induce Lys48 polyubiquitination and proteasomal degradation of the target protein.

Fig. 4 .
Fig. 4. Schematic representation of neuron-glia interaction-based drug development.For the establishment of biomarkers assisting tauopathy-targeting therapies, we propose a workflow consisting of the following elements: (1) Microglia are isolated from WT and tauopathy model mice at different ages for single-cell RNA-seq gene expression profiling.(2) The DAM signature in tauopathy mouse models is examined for staging the tau-related neurodegenerative phenotype of microglia, and candidates of neuroinflammatory biomarkers can be selected from stage-dependent gene expressions.(3) Microglia pathological stages can be linked to tau pathology stages defined by tau PET imaging for further selection of tauopathy-related DAM markers, and this approach can be supplemented by immunohistochemical and biochemical assays of excised tissues.(4) Selective small-molecule, brain-entering PET radioligands for the selected molecular targets are developed by in-vivo imaging and in-vitro autoradiographic evaluations.PET images of tau pathologies and neuroinflammatory changes in this figure tentatively illustrate the performance of 18 F-PM-PBB3 and translocator protein (TSPO) tracer,11 C-Ac5216, respectively, in rTg4510 mouse brains putatively at three different stages.However, new neuroinflammatory PET ligands will need to be generated as TSPO is not included in DAM makers.

Table 1 .
Properties of tau PET tracers.Relative performance of each radioprobe was indicated according to contrasts for AD tau lesions.Some of radioprobes detected in vivo tau pathology of non-AD tauopathies.Monoamine oxidase (MAO)-A, MAO-B and choroid plexus (CP) are known as off-targets of several tau PET tracers.