A microtubule stabilizer ameliorates protein pathogenesis and neurodegeneration in mouse models of repetitive traumatic brain injury.

Tau pathogenesis is a hallmark of many neurodegenerative diseases, including Alzheimer's disease (AD). Although the events leading to initial tau misfolding and subsequent tau spreading in patient brains are largely unknown, traumatic brain injury (TBI) may be a risk factor for tau-mediated neurodegeneration. Using a repetitive TBI (rTBI) paradigm, we report that rTBI induced somatic accumulation of phosphorylated and misfolded tau, as well as neurodegeneration across multiple brain areas in 7-month-old tau transgenic PS19 mice but not wild-type (WT) mice. rTBI accelerated somatic tau pathology in younger PS19 mice and WT mice only after inoculation with tau preformed fibrils and AD brain-derived pathological tau (AD-tau), respectively, suggesting that tau seeds are needed for rTBI-induced somatic tau pathology. rTBI further disrupted axonal microtubules and induced punctate tau and TAR DNA binding protein 43 (TDP-43) pathology in the optic tracts of WT mice. These changes in the optic tract were associated with a decline of visual function. Treatment with a brain-penetrant microtubule-stabilizing molecule reduced rTBI-induced tau, TDP-43 pathogenesis, and neurodegeneration in the optic tract as well as visual dysfunction. Treatment with the microtubule stabilizer also alleviated rTBI-induced tau pathology in the cortices of AD-tau-inoculated WT mice. These results indicate that rTBI facilitates abnormal microtubule organization, pathological tau formation, and neurodegeneration and suggest microtubule stabilization as a potential therapeutic avenue for TBI-induced neurodegeneration.

Inhibition of CK2 mitigates Alzheimer's tau pathology by preventing NR2B synaptic mislocalization.

Alzheimer's disease (AD) is a neurodegenerative disorder that exhibits pathological changes in both tau and synaptic function. AD patients display increases in hyperphosphorylated tau and synaptic activity. Previous studies have individually identified the role of NR2B subunit-containing NMDA receptors in AD related synaptic dysfunction and aggregated tau without reconciling the conflicting differences and implications of NR2B expression. Inhibition of extrasynaptically located NR2B mitigates tau pathology in AD models, whereas the inhibition of synaptic NR2B replicates tau-associated hyperactivity. This suggests that a simultaneous increase in extrasynaptic NR2B and decrease in synaptic NR2B may be responsible for tau pathology and synaptic dysfunction, respectively. The synaptic location of NR2B is regulated by casein kinase 2 (CK2), which is highly expressed in AD patients. Here, we used patient brains diagnosed with AD, corticobasal degeneration, progressive supranuclear palsy or Pick's disease to characterize CK2 expression across these diverse tauopathies. Human derived material was also utilized in conjunction with cultured hippocampal neurons in order to investigate AD-induced changes in NR2B location. We further assessed the therapeutic effect of CK2 inhibition on NR2B synaptic distribution and tau pathology. We found that aberrant expression of CK2, and synaptically translocated NR2B, is unique to AD patients compared to other tauopathies. Increased CK2 was also observed in AD-tau treated neurons in addition to the mislocalization of NR2B receptors. Tau burden was alleviated in vitro by correcting synaptic:extrasynaptic NR2B function. Restoring NR2B physiological expression patterns with CK2 inhibition and inhibiting the function of excessive extrasynaptic NR2B with Memantine both mitigated tau accumulation in vitro. However, the combined pharmacological treatment promoted the aggregation of tau. Our data suggests that the synaptic:extrasynaptic balance of NR2B function regulates AD-tau pathogenesis, and that the inhibition of CK2, and concomitant prevention of NR2B mislocalization, may be a useful therapeutic tool for AD patients.

Distinct characteristics of limbic-predominant age-related TDP-43 encephalopathy in Lewy body disease.

Limbic-predominant age-related TDP-43 encephalopathy (LATE) is characterized by the accumulation of TAR-DNA-binding protein 43 (TDP-43) aggregates in older adults. LATE coexists with Lewy body disease (LBD) as well as other neuropathological changes including Alzheimer's disease (AD). We aimed to identify the pathological, clinical, and genetic characteristics of LATE in LBD (LATE-LBD) by comparing it with LATE in AD (LATE-AD), LATE with mixed pathology of LBD and AD (LATE-LBD + AD), and LATE alone (Pure LATE). We analyzed four cohorts of autopsy-confirmed LBD (n = 313), AD (n = 282), LBD + AD (n = 355), and aging (n = 111). We assessed the association of LATE with patient profiles including LBD subtype and AD neuropathologic change (ADNC). We studied the morphological and distributional differences between LATE-LBD and LATE-AD. By frequency analysis, we staged LATE-LBD and examined the association with cognitive impairment and genetic risk factors. Demographic analysis showed LATE associated with age in all four cohorts and the frequency of LATE was the highest in LBD + AD followed by AD, LBD, and Aging. LBD subtype and ADNC associated with LATE in LBD or AD but not in LBD + AD. Pathological analysis revealed that the hippocampal distribution of LATE was different between LATE-LBD and LATE-AD: neuronal cytoplasmic inclusions were more frequent in cornu ammonis 3 (CA3) in LATE-LBD compared to LATE-AD and abundant fine neurites composed of C-terminal truncated TDP-43 were found mainly in CA2 to subiculum in LATE-LBD, which were not as numerous in LATE-AD. Some of these fine neurites colocalized with phosphorylated α-synuclein. LATE-LBD staging showed LATE neuropathological changes spread in the dentate gyrus and brainstem earlier than in LATE-AD. The presence and prevalence of LATE in LBD associated with cognitive impairment independent of either LBD subtype or ADNC; LATE-LBD stage also associated with the genetic risk variants of TMEM106B rs1990622 and GRN rs5848. These data highlight clinicopathological and genetic features of LATE-LBD.

In vitro amplification of pathogenic tau conserves disease-specific bioactive characteristics.

The microtubule-associated protein tau (tau) forms hyperphosphorylated aggregates in the brains of tauopathy patients that can be pathologically and biochemically defined as distinct tau strains. Recent studies show that these tau strains exhibit strain-specific biological activities, also referred to as pathogenicities, in the tau spreading models. Currently, the specific pathogenicity of human-derived tau strains cannot be fully recapitulated by synthetic tau preformed fibrils (pffs), which are generated from recombinant tau protein. Reproducing disease-relevant tau pathology in cell and animal models necessitates the use of human brain-derived tau seeds. However, the availability of human-derived tau is extremely limited. Generation of tau variants that can mimic the pathogenicity of human-derived tau seeds would significantly extend the scale of experimental design within the field of tauopathy research. Previous studies have demonstrated that in vitro seeding reactions can amplify the beta-sheet structure of tau protein from a minute quantity of human-derived tau. However, whether the strain-specific pathogenicities of the original, human-derived tau seeds are conserved in the amplified tau strains has yet to be experimentally validated. Here, we used biochemically enriched brain-derived tau seeds from Alzheimer's disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) patient brains with a modified seeding protocol to template the recruitment of recombinant 2N4R (T40) tau in vitro. We quantitatively interrogated efficacy of the amplification reactions and the pathogenic fidelity of the amplified material to the original tau seeds using recently developed sporadic tau spreading models. Our data suggest that different tau strains can be faithfully amplified in vitro from tau isolated from different tauopathy brains and that the amplified tau variants retain their strain-dependent pathogenic characteristics.

Insoluble Tau From Human FTDP-17 Cases Exhibit Unique Transmission Properties In Vivo.

One hallmark of neurodegenerative diseases is the intracellular accumulation of hyperphosphorylated tau protein, a neuronal microtubule-associated protein, into structures known as neurofibrillary tangles. Tauopathies are heterogeneous neurodegenerative diseases caused by the misfolding of the tau protein. It has been previously shown that the tau protein can spread from cell to cell in a prion-like manner. Tauopathies can be sporadic or familial, with the identification of pathogenic mutations in the microtubule-associated protein tau gene on chromosome 17 in the familial cases. Different frontotemporal dementia with parkinsonism-17 (FTDP-17) cases are associated with varying clinical presentations and types of neuropathology. We previously demonstrated that insoluble tau extracted from sporadic tauopathy human brains contain distinct tau strains, which underlie the heterogeneity of these diseases. Furthermore, these tau strains seeded tau aggregates that resemble human tau neuropathology in nontransgenic and 6hTau mice in vivo. Here, we show insoluble tau from human brains of FTDP-17 cases transmit different patterns of neuronal and glial tau pathology in vivo, similar to the sporadic tauopathies. This suggests that each of these tau mutations has unique properties that underlie the heterogeneity of FTDP-17 cases.

Transmission of tauopathy strains is independent of their isoform composition.

The deposition of pathological tau is a common feature in several neurodegenerative tauopathies. Although equal ratios of tau isoforms with 3 (3R) and 4 (4R) microtubule-binding repeats are expressed in the adult human brain, the pathological tau from different tauopathies have distinct isoform compositions and cell type specificities. The underlying mechanisms of tauopathies are unknown, partially due to the lack of proper models. Here, we generate a new transgenic mouse line expressing equal ratios of 3R and 4R human tau isoforms (6hTau mice). Intracerebral injections of distinct human tauopathy brain-derived tau strains into 6hTau mice recapitulate the deposition of pathological tau with distinct tau isoform compositions and cell type specificities as in human tauopathies. Moreover, through in vivo propagation of these tau strains among different mouse lines, we demonstrate that the transmission of distinct tau strains is independent of strain isoform compositions, but instead intrinsic to unique pathological conformations.

Amyloid-ß plaques enhance Alzheimer's brain tau-seeded pathologies by facilitating neuritic plaque tau aggregation.

Alzheimer's disease (AD) is characterized by extracellular amyloid-ß (Aß) plaques and intracellular tau inclusions. However, the exact mechanistic link between these two AD lesions remains enigmatic. Through injection of human AD-brain-derived pathological tau (AD-tau) into Aß plaque-bearing mouse models that do not overexpress tau, we recapitulated the formation of three major types of AD-relevant tau pathologies: tau aggregates in dystrophic neurites surrounding Aß plaques (NP tau), AD-like neurofibrillary tangles (NFTs) and neuropil threads (NTs). These distinct tau pathologies have different temporal onsets and functional consequences on neural activity and behavior. Notably, we found that Aß plaques created a unique environment that facilitated the rapid amplification of proteopathic AD-tau seeds into large tau aggregates, initially appearing as NP tau, which was followed by the formation and spread of NFTs and NTs, likely through secondary seeding events. Our study provides insights into a new multistep mechanism underlying Aß plaque-associated tau pathogenesis.

Pathological Tau Strains from Human Brains Recapitulate the Diversity of Tauopathies in Nontransgenic Mouse Brain.

Pathological tau aggregates occur in Alzheimer's disease (AD) and other neurodegenerative tauopathies. It is not clearly understood why tauopathies vary greatly in the neuroanatomical and histopathological patterns of tau aggregation, which contribute to clinical heterogeneity in these disorders. Recent studies have shown that tau aggregates may form distinct structural conformations, known as tau strains. Here, we developed a novel model to test the hypothesis that cell-to-cell transmission of different tau strains occurs in nontransgenic (non-Tg) mice, and to investigate whether there are strain-specific differences in the pattern of tau transmission. By injecting pathological tau extracted from postmortem brains of AD (AD-tau), progressive supranuclear palsy (PSP-tau), and corticobasal degeneration (CBD-tau) patients into different brain regions of female non-Tg mice, we demonstrated the induction and propagation of endogenous mouse tau aggregates. Specifically, we identified differences in tau strain potency between AD-tau, CBD-tau, and PSP-tau in non-Tg mice. Moreover, differences in cell-type specificity of tau aggregate transmission were observed between tau strains such that only PSP-tau and CBD-tau strains induce astroglial and oligodendroglial tau inclusions, recapitulating the diversity of neuropathology in human tauopathies. Furthermore, we demonstrated that the neuronal connectome, but not the tau strain, determines which brain regions develop tau pathology. Finally, CBD-tau- and PSP-tau-injected mice showed spatiotemporal transmission of glial tau pathology, suggesting glial tau transmission contributes to the progression of tauopathies. Together, our data suggest that different tau strains determine seeding potency and cell-type specificity of tau aggregation that underlie the diversity of human tauopathies.SIGNIFICANCE STATEMENT Tauopathies show great clinical and neuropathological heterogeneity, despite the fact that tau aggregates in each disease. This heterogeneity could be due to tau aggregates forming distinct structural conformations, or strains. We now report the development of a sporadic tauopathy model to study human tau strains by intracerebrally injecting nontransgenic mice with pathological tau enriched from human tauopathy brains. We show human tau strains seed different types and cellular distributions of tau neuropathology in our model that recapitulate the heterogeneity seen in these human diseases.

Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis.

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G4C2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G4C2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72-associated ALS (c9ALS). Therapeutics that target G4C2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G4C2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G4C2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G4C2 RNA and downstream G4C2 RNA-mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G4C2 RNA-based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention.

Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice.

Filamentous tau aggregates are hallmark lesions in numerous neurodegenerative diseases, including Alzheimer's disease (AD). Cell culture and animal studies showed that tau fibrils can undergo cell-to-cell transmission and seed aggregation of soluble tau, but this phenomenon was only robustly demonstrated in models overexpressing tau. In this study, we found that intracerebral inoculation of tau fibrils purified from AD brains (AD-tau), but not synthetic tau fibrils, resulted in the formation of abundant tau inclusions in anatomically connected brain regions in nontransgenic mice. Recombinant human tau seeded by AD-tau revealed unique conformational features that are distinct from synthetic tau fibrils, which could underlie the differential potency in seeding physiological levels of tau to aggregate. Therefore, our study establishes a mouse model of sporadic tauopathies and points to important differences between tau fibrils that are generated artificially and authentic ones that develop in AD brains.

Tau pathology spread in PS19 tau transgenic mice following locus coeruleus (LC) injections of synthetic tau fibrils is determined by the LC's afferent and efferent connections.

Filamentous tau inclusions are hallmarks of Alzheimer's disease (AD) and other neurodegenerative tauopathies. An increasing number of studies implicate the cell-to-cell propagation of tau pathology in the progression of tauopathies. We recently showed (Iba et al., J Neurosci 33:1024-1037, 2013) that inoculation of preformed synthetic tau fibrils (tau PFFs) into the hippocampus of young transgenic (Tg) mice (PS19) overexpressing human P301S mutant tau induced robust tau pathology in anatomically connected brain regions including the locus coeruleus (LC). Since Braak and colleagues hypothesized that the LC is the first brain structure to develop tau lesions and since LC has widespread connections throughout the CNS, LC neurons could be the critical initiators of the stereotypical spreading of tau pathology through connectome-dependent transmission of pathological tau in AD. Here, we report that injections of tau PFFs into the LC of PS19 mice induced propagation of tau pathology to major afferents and efferents of the LC. Notably, tau pathology propagated along LC efferent projections was localized not only to axon terminals but also to neuronal perikarya, suggesting transneuronal transfer of templated tau pathology to neurons receiving LC projections. Further, brainstem neurons giving rise to major LC afferents also developed perikaryal tau pathology. Surprisingly, while tangle-bearing neurons degenerated in the LC ipsilateral to the injection site starting 6 months post-injection, no neuron loss was seen in the contralateral LC wherein tangle-bearing neurons gradually cleared tau pathology by 6-12 months post-injection. However, the spreading pattern of tau pathology observed in our LC-injected mice is different from that in AD brains since hippocampus and entorhinal cortex, which are affected in early stages of AD, were largely spared of tau inclusions in our model. Thus, while our study tested critical aspects of the Braak hypothesis of tau pathology spread, this novel mouse model provides unique opportunities to elucidate mechanisms underlying the selective vulnerability of neurons to acquire tau pathology and succumb to or resist tau-mediated neurodegeneration.

Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer's-like tauopathy.

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau pathologies, are neurodegenerative diseases characterized by neurofibrillary tangles (NFTs) comprising filamentous tau protein. Although emerging evidence suggests that tau pathology may be transmitted, we demonstrate here that synthetic tau fibrils are sufficient to transmit tau inclusions in a mouse model. Specifically, intracerebral inoculation of young PS19 mice overexpressing mutant human tau (P301S) with synthetic preformed fibrils (pffs) assembled from recombinant full-length tau or truncated tau containing four microtubule binding repeats resulted in rapid induction of NFT-like inclusions that propagated from injected sites to connected brain regions in a time-dependent manner. Interestingly, injection of tau pffs into either hippocampus or striatum together with overlaying cortex gave rise to distinct pattern of spreading. Moreover, unlike tau pathology that spontaneously develops in old PS19 mice, the pff-induced tau inclusions more closely resembled AD NFTs because they were Thioflavin S positive, acetylated, and more resistant to proteinase K digestion. Together, our study demonstrates that synthetic tau pffs alone are capable of inducing authentic NFT-like tau aggregates and initiating spreading of tau pathology in a tauopathy mouse model.