Immunotherapy with an antibody against CD1d modulates neuroinflammation in an α-synuclein transgenic model of Lewy body like disease.

The neuroinflammatory process in synucleinopathies of the aging population such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) involves microglial activation as well as infiltration of the CNS by T cells and natural killer T cells (NKTs). To evaluate the potential of targeting NKT cells to modulate neuroinflammation, we treated α-syn transgenic (tg) mice (e.g.: Thy1 promoter line 61) with an antibody against CD1d, which is a glycoprotein expressed in antigen presenting cells (APCs). CD1d-presented lipid antigens activate NKT cells through the interaction with T cell receptor in NKTs, resulting in the production of cytokines. Thus, we hypothesized that blocking the APC-NKT interaction with an anti-CD1d antibody might reduce neuroinflammation and neurodegeneration in models of DLB/PD. Treatment with the anti-CD1d antibody did not have effects on CD3 (T cells), slightly decreased CD4 and increased CD8 lymphocytes in the mice. Moreover, double labeling studies showed that compared to control (IgG) treated α-syn tg mice, treatment with anti-CD1d decreased numbers of CD3/interferon γ (IFN γ)-positive cells, consistent with NKTs. Further double labeling studies showed that CD1d-positive cells co-localized with the astrocytes marker GFAP and that anti-CD1d antibody reduced this effect. While in control α-syn tg mice CD3 positive cells were near astrocytes, this was modified by the treatment with the CD1d antibody. By qPCR, levels of IFN γ, CCL4, and interleukin-6 were increased in the IgG treated α-syn tg mice. Treatment with CD1d antibody blunted this cytokine response that was associated with reduced astrocytosis and microgliosis in the CNS of the α-syn tg mice treated with CD1d antibody. Flow cytometric analysis of immune cells in α-syn tg mice revealed that CD1d-tet + T cells were also increased in the spleen of α-syn tg mice, which treatment with the CD1d antibody reduced. Reduced neuroinflammation in the anti-CD1d-treated α-syn tg mice was associated with amelioration of neurodegenerative pathology. These results suggest that reducing infiltration of NKT cells with an antibody against CD1d might be a potential therapeutical approach for DLB/PD.

Inhibition of p38α MAPK restores neuronal p38γ MAPK and ameliorates synaptic degeneration in a mouse model of DLB/PD.

Alterations in the p38 mitogen-activated protein kinases (MAPKs) play an important role in the pathogenesis of dementia with Lewy bodies (DLB) and Parkinson's disease (PD). Activation of the p38α MAPK isoform and mislocalization of the p38γ MAPK isoform are associated with neuroinflammation and synaptic degeneration in DLB and PD. Therefore, we hypothesized that p38α might be associated with neuronal p38γ distribution and synaptic dysfunction in these diseases. To test this hypothesis, we treated in vitro cellular and in vivo mouse models of DLB/PD with SKF-86002, a compound that attenuates inflammation by inhibiting p38α/ß, and then investigated the effects of this compound on p38γ and neurodegenerative pathology. We found that inhibition of p38α reduced neuroinflammation and ameliorated synaptic, neurodegenerative, and motor behavioral deficits in transgenic mice overexpressing human α-synuclein. Moreover, treatment with SKF-86002 promoted the redistribution of p38γ to synapses and reduced the accumulation of α-synuclein in mice overexpressing human α-synuclein. Supporting the potential value of targeting p38 in DLB/PD, we found that SKF-86002 promoted the redistribution of p38γ in neurons differentiated from iPS cells derived from patients with familial PD (carrying the A53T α-synuclein mutation) and healthy controls. Treatment with SKF-86002 ameliorated α-synuclein-induced neurodegeneration in these neurons only when microglia were pretreated with this compound. However, direct treatment of neurons with SKF-86002 did not affect α-synuclein-induced neurotoxicity, suggesting that SKF-86002 treatment inhibits α-synuclein-induced neurotoxicity mediated by microglia. These findings provide a mechanistic connection between p38α and p38γ as well as a rationale for targeting this pathway in DLB/PD.

Specific Detection of Physiological S129 Phosphorylated α-Synuclein in Tissue Using Proximity Ligation Assay.

BACKGROUND: Synucleinopathies are a group of neurodegenerative disorders that are pathologically characterized by intracellular aggregates called Lewy bodies. Lewy bodies are primarily composed of α-synuclein (asyn) protein, which is mostly phosphorylated at serine 129 (pS129) when aggregated and therefore used as a marker for pathology. Currently commercial antibodies against pS129 asyn stain aggregates well but in healthy brains cross react with other proteins, thus making it difficult to specifically detect physiological pS129 asyn. OBJECTIVE: To develop a staining procedure that detects endogenous and physiological relevant pS129 asyn with high specificity and low background. METHODS: We used the fluorescent and brightfield in situ proximity ligation assay (PLA) to specifically detect pS129 asyn in cell culture, mouse, and human brain sections. RESULTS: The pS129 asyn PLA specifically stained physiological and soluble pS129 asyn in cell culture, mouse brain sections, and human brain tissue without significant cross-reactivity or background signal. However, this technique was not successful in detecting Lewy bodies in human brain tissue. CONCLUSION: We successfully developed a novel PLA method that can, in the future, be used on in vitro and in vivo samples as a tool to explore and better understand the cellular localization and function of pS129 asyn in health and disease.

Aging exacerbates the brain inflammatory micro-environment contributing to α-synuclein pathology and functional deficits in a mouse model of DLB/PD.

BACKGROUND: Although ɑ-synuclein (ɑ-syn) spreading in age-related neurodegenerative diseases such as Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) has been extensively investigated, the role of aging in the manifestation of disease remains unclear. METHODS: We explored the role of aging and inflammation in the pathogenesis of synucleinopathies in a mouse model of DLB/PD initiated by intrastriatal injection of ɑ-syn preformed fibrils (pff). RESULTS: We found that aged mice showed more extensive accumulation of ɑ-syn in selected brain regions and behavioral deficits that were associated with greater infiltration of T cells and microgliosis. Microglial inflammatory gene expression induced by ɑ-syn-pff injection in young mice had hallmarks of aged microglia, indicating that enhanced age-associated pathologies may result from inflammatory synergy between aging and the effects of ɑ-syn aggregation. Based on the transcriptomics analysis projected from Ingenuity Pathway Analysis, we found a network that included colony stimulating factor 2 (CSF2), LPS related genes, TNFɑ and poly rl:rC-RNA as common regulators. CONCLUSIONS: We propose that aging related inflammation (eg: CSF2) influences outcomes of pathological spreading of ɑ-syn and suggest that targeting neuro-immune responses might be important in developing treatments for DLB/PD.

Does SARS-CoV-2 affect neurodegenerative disorders? TLR2, a potential receptor for SARS-CoV-2 in the CNS.

The coronavirus (COVID-19) pandemic, caused by severe acute respiratory system coronavirus 2 (SARS-CoV-2), has created significant challenges for scientists seeking to understand the pathogenic mechanisms of SARS-CoV-2 infection and to identify the best therapies for infected patients. Although ACE2 is a known receptor for the virus and has been shown to mediate viral entry into the lungs, accumulating reports highlight the presence of neurological symptoms resulting from infection. As ACE2 expression is low in the central nervous system (CNS), these neurological symptoms are unlikely to be caused by ACE2-virus binding. In this review, we will discuss a proposed interaction between SARS-CoV-2 and Toll-like receptor 2 (TLR2) in the CNS. TLR2 is an innate immune receptor that recognizes exogenous microbial components but has also been shown to interact with multiple viral components, including the envelope (E) protein of SARS-CoV-2. In addition, TLR2 plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Based on these observations, we hypothesize that TLR2 may play a critical role in the response to SARS-CoV-2 infiltration in the CNS, thereby resulting in the induction or acceleration of AD and PD pathologies in patients.

Efficacy and immunogenicity of MultiTEP-based DNA vaccines targeting human α-synuclein: prelude for IND enabling studies.

Accumulation of misfolded proteins such as amyloid-ß (Aß), tau, and α-synuclein (α-Syn) in the brain leads to synaptic dysfunction, neuronal damage, and the onset of relevant neurodegenerative disorder/s. Dementia with Lewy bodies (DLB) and Parkinson's disease (PD) are characterized by the aberrant accumulation of α-Syn intracytoplasmic Lewy body inclusions and dystrophic Lewy neurites resulting in neurodegeneration associated with inflammation. Cell to cell propagation of α-Syn aggregates is implicated in the progression of PD/DLB, and high concentrations of anti-α-Syn antibodies could inhibit/reduce the spreading of this pathological molecule in the brain. To ensure sufficient therapeutic concentrations of anti-α-Syn antibodies in the periphery and CNS, we developed four α-Syn DNA vaccines based on the universal MultiTEP platform technology designed especially for the elderly with immunosenescence. Here, we are reporting on the efficacy and immunogenicity of these vaccines targeting three B-cell epitopes of hα-Syn aa85-99 (PV-1947D), aa109-126 (PV-1948D), aa126-140 (PV-1949D) separately or simultaneously (PV-1950D) in a mouse model of synucleinopathies mimicking PD/DLB. All vaccines induced high titers of antibodies specific to hα-Syn that significantly reduced PD/DLB-like pathology in hα-Syn D line mice. The most significant reduction of the total and protein kinase resistant hα-Syn, as well as neurodegeneration, were observed in various brain regions of mice vaccinated with PV-1949D and PV-1950D in a sex-dependent manner. Based on these preclinical data, we selected the PV-1950D vaccine for future IND enabling preclinical studies and clinical development.

Effects of innate immune receptor stimulation on extracellular α-synuclein uptake and degradation by brain resident cells.

Synucleinopathies are age-related neurological disorders characterized by the progressive deposition of α-synuclein (α-syn) aggregates and include Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Although cell-to-cell α-syn transmission is thought to play a key role in the spread of α-syn pathology, the detailed mechanism is still unknown. Neuroinflammation is another key pathological feature of synucleinopathies. Previous studies have identified several immune receptors that mediate neuroinflammation in synucleinopathies, such as Toll-like receptor 2 (TLR2). However, the species of α-syn aggregates varies from study to study, and how different α-syn aggregate species interact with innate immune receptors has yet to be addressed. Therefore, we investigated whether innate immune receptors can facilitate the uptake of different species of α-syn aggregates. Here, we examined whether stimulation of TLRs could modulate the cellular uptake and degradation of α-syn fibrils despite a lack of direct interaction. We observed that stimulation of TLR2 in vitro accelerated α-syn fibril uptake in neurons and glia while delaying the degradation of α-syn in neurons and astrocytes. Internalized α-syn was rapidly degraded in microglia regardless of whether TLR2 was stimulated. However, cellular α-syn uptake and degradation kinetics were not altered by TLR4 stimulation. In addition, upregulation of TLR2 expression in a synucleinopathy mouse model increased the density of Lewy-body-like inclusions and induced morphological changes in microglia. Together, these results suggest that cell type-specific modulation of TLR2 may be a multifaceted and promising therapeutic strategy for synucleinopathies; inhibition of neuronal and astroglial TLR2 decreases pathogenic α-syn transmission, but activation of microglial TLR2 enhances microglial extracellular α-syn clearance.

LRRK2 mediates microglial neurotoxicity via NFATc2 in rodent models of synucleinopathies.

Synucleinopathies are neurodegenerative disorders characterized by abnormal α-synuclein deposition that include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The pathology of these conditions also includes neuronal loss and neuroinflammation. Neuron-released α-synuclein has been shown to induce neurotoxic, proinflammatory microglial responses through Toll-like receptor 2, but the molecular mechanisms involved are poorly understood. Here, we show that leucine-rich repeat kinase 2 (LRRK2) plays a critical role in the activation of microglia by extracellular α-synuclein. Exposure to α-synuclein was found to enhance LRRK2 phosphorylation and activity in mouse primary microglia. Furthermore, genetic and pharmacological inhibition of LRRK2 markedly diminished α-synuclein-mediated microglial neurotoxicity via lowering of tumor necrosis factor-α and interleukin-6 expression in mouse cultures. We determined that LRRK2 promoted a neuroinflammatory cascade by selectively phosphorylating and inducing nuclear translocation of the immune transcription factor nuclear factor of activated T cells, cytoplasmic 2 (NFATc2). NFATc2 activation was seen in patients with synucleinopathies and in a mouse model of synucleinopathy, where administration of an LRRK2 pharmacological inhibitor restored motor behavioral deficits. Our results suggest that modulation of LRRK2 and its downstream signaling mediator NFATc2 might be therapeutic targets for treating synucleinopathies.

Neuroinflammation is associated with infiltration of T cells in Lewy body disease and α-synuclein transgenic models.

BACKGROUND: α-Synuclein (α-syn) is a pre-synaptic protein which progressively accumulates in neuronal and non-neuronal cells in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy. Recent evidence suggests that aberrant immune activation may be involved in neurodegeneration in PD/DLB. While previous studies have often focused on the microglial responses, less is known about the role of the peripheral immune system in these disorders. METHODS: To understand the involvement of the peripheral immune system in PD/DLB, we evaluated T cell populations in the brains of α-syn transgenic (tg) mice (e.g., Thy1 promoter line 61) and DLB patients. RESULTS: Immunohistochemical analysis showed perivascular and parenchymal infiltration by CD3+/CD4+ helper T cells, but not cytotoxic T cells (CD3+/CD8+) or B cells (CD20+), in the neocortex, hippocampus, and striatum of α-syn tg mice. CD3+ cells were found in close proximity to the processes of activated astroglia, particularly in areas of the brain with significant astrogliosis, microgliosis, and expression of pro-inflammatory cytokines. In addition, a subset of CD3+ cells co-expressed interferon γ. Flow cytometric analysis of immune cells in the brains of α-syn tg mice revealed that CD1d-tet+ T cells were also increased in the brains of α-syn tg mice suggestive of natural killer T cells. In post-mortem DLB brains, we similarly detected increased numbers of infiltrating CD3+/CD4+ T cells in close proximity with blood vessels. CONCLUSION: These results suggest that infiltrating adaptive immune cells play an important role in neuroinflammation and neurodegeneration in synucleinopathies and that modulating peripheral T cells may be a viable therapeutic strategy for PD/DLB.

Role of Alterations in Protein Kinase p38γ in the Pathogenesis of the Synaptic Pathology in Dementia With Lewy Bodies and α-Synuclein Transgenic Models.

Progressive accumulation of the pre-synaptic protein α-synuclein (α-syn) has been strongly associated with the pathogenesis of neurodegenerative disorders of the aging population such as Alzheimer's disease (AD), Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy. While the precise mechanisms are not fully understood, alterations in kinase pathways including that of mitogen activated protein kinase (MAPK) p38 have been proposed to play a role. In AD, p38α activation has been linked to neuro-inflammation while alterations in p38γ have been associated with tau phosphorylation. Although p38 has been studied in AD, less is known about its role in DLB/PD and other α-synucleinopathies. For this purpose, we investigated the expression of the p38 family in brains from α-syn overexpressing transgenic mice (α-syn Tg: Line 61) and patients with DLB/PD. Immunohistochemical analysis revealed that in healthy human controls and non-Tg mice, p38α associated with neurons and astroglial cells and p38γ localized to pre-synaptic terminals. In DLB and α-syn Tg brains, however, p38α levels were increased in astroglial cells while p38γ immunostaining was redistributed from the synaptic terminals to the neuronal cell bodies. Double immunolabeling further showed that p38γ colocalized with α-syn aggregates in DLB patients, and immunoblot and qPCR analysis confirmed the increased levels of p38α and p38γ. α1-syntrophin, a synaptic target of p38γ, was present in the neuropil and some neuronal cell bodies in human controls and non-Tg mice. In DLB and and Tg mice, however, α1-syntrophin was decreased in the neuropil and instead colocalized with α-syn in intra-neuronal inclusions. In agreement with these findings, in vitro studies showed that α-syn co-immunoprecipitates with p38γ, but not p38α. These results suggest that α-syn might interfere with the p38γ pathway and play a role in the mechanisms of synaptic dysfunction in DLB/PD.

Immunotherapies for Aging-Related Neurodegenerative Diseases-Emerging Perspectives and New Targets.

Neurological disorders such as Alzheimer's disease (AD), Lewy body dementia (LBD), frontotemporal dementia (FTD), and vascular dementia (VCID) have no disease-modifying treatments to date and now constitute a dementia crisis that affects 5 million in the USA and over 50 million worldwide. The most common pathological hallmark of these age-related neurodegenerative diseases is the accumulation of specific proteins, including amyloid beta (Aß), tau, α-synuclein (α-syn), TAR DNA-binding protein 43 (TDP43), and repeat-associated non-ATG (RAN) peptides, in the intra- and extracellular spaces of selected brain regions. Whereas it remains controversial whether these accumulations are pathogenic or merely a byproduct of disease, the majority of therapeutic research has focused on clearing protein aggregates. Immunotherapies have garnered particular attention for their ability to target specific protein strains and conformations as well as promote clearance. Immunotherapies can also be neuroprotective: by neutralizing extracellular protein aggregates, they reduce spread, synaptic damage, and neuroinflammation. This review will briefly examine the current state of research in immunotherapies against the 3 most commonly targeted proteins for age-related neurodegenerative disease: Aß, tau, and α-syn. The discussion will then turn to combinatorial strategies that enhance the effects of immunotherapy against aggregating protein, followed by new potential targets of immunotherapy such as aging-related processes.

Targeting Microglial and Neuronal Toll-like Receptor 2 in Synucleinopathies.

Synucleinopathies are neurodegenerative disorders characterized by the progressive accumulation of α-synuclein (α-syn) in neurons and glia and include Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In this review, we consolidate our key findings and recent studies concerning the role of Toll-like receptor 2 (TLR2), a pattern recognition innate immune receptor, in the pathogenesis of synucleinopathies. First, we address the pathological interaction of α-syn with microglial TLR2 and its neurotoxic inflammatory effects. Then, we show that neuronal TLR2 activation not only induces abnormal α-syn accumulation by impairing autophagy, but also modulates α-syn transmission. Finally, we demonstrate that administration of a TLR2 functional inhibitor improves the neuropathology and behavioral deficits of a synucleinopathy mouse model. Altogether, we present TLR2 modulation as a promising immunotherapy for synucleinopathies.

Immunotherapy targeting toll-like receptor 2 alleviates neurodegeneration in models of synucleinopathy by modulating α-synuclein transmission and neuroinflammation.

BACKGROUND: Synucleinopathies of the aging population are an heterogeneous group of neurological disorders that includes Parkinson's disease (PD) and dementia with Lewy bodies (DLB) and are characterized by the progressive accumulation of α-synuclein in neuronal and glial cells. Toll-like receptor 2 (TLR2), a pattern recognition immune receptor, has been implicated in the pathogenesis of synucleinopathies because TLR2 is elevated in the brains of patients with PD and TLR2 is a mediator of the neurotoxic and pro-inflammatory effects of extracellular α-synuclein aggregates. Therefore, blocking TLR2 might alleviate α-synuclein pathological and functional effects. For this purpose, herein, we targeted TLR2 using a functional inhibitory antibody (anti-TLR2). METHODS: Two different human α-synuclein overexpressing transgenic mice were used in this study. α-synuclein low expresser mouse (α-syn-tg, under the PDGFß promoter, D line) was stereotaxically injected with TLR2 overexpressing lentivirus to demonstrate that increment of TLR2 expression triggers neurotoxicity and neuroinflammation. α-synuclein high expresser mouse (α-Syn-tg; under mThy1 promoter, Line 61) was administrated with anti-TLR2 to examine that functional inhibition of TLR2 ameliorates neuropathology and behavioral defect in the synucleinopathy animal model. In vitro α-synuclein transmission live cell monitoring system was used to evaluate the role of TLR2 in α-synuclein cell-to-cell transmission. RESULTS: We demonstrated that administration of anti-TLR2 alleviated α-synuclein accumulation in neuronal and astroglial cells, neuroinflammation, neurodegeneration, and behavioral deficits in an α-synuclein tg mouse model of PD/DLB. Moreover, in vitro studies with neuronal and astroglial cells showed that the neuroprotective effects of anti-TLR2 antibody were mediated by blocking the neuron-to-neuron and neuron-to-astrocyte α-synuclein transmission which otherwise promotes NFκB dependent pro-inflammatory responses. CONCLUSION: This study proposes TLR2 immunotherapy as a novel therapeutic strategy for synucleinopathies of the aging population.

Longitudinal imaging reveals subhippocampal dynamics in glutamate levels associated with histopathologic events in a mouse model of tauopathy and healthy mice.

Tauopathies are neurodegenerative disorders characterized by abnormal intracellular aggregates of tau protein, and include Alzheimer's disease, corticobasal degeneration, frontotemporal dementia, and traumatic brain injury. Glutamate metabolism is altered in neurodegenerative disorders manifesting in higher or lower concentrations of glutamate, its transporters or receptors. Previously, glutamate chemical exchange saturation transfer (GluCEST) magnetic resonance imaging (MRI) demonstrated that glutamate levels are reduced in regions of synapse loss in the hippocampus of a mouse model of late-stage tauopathy. We performed a longitudinal GluCEST imaging experiment paired with a cross-sectional study of histologic markers of tauopathy to determine whether (1) early GluCEST changes are associated with synapse loss before volume loss occurs in the hippocampus, and whether (2) subhippocampal dynamics in GluCEST are associated with histopathologic events related to glutamate alterations in tauopathy. Live imaging of the hippocampus in three serial slices was performed without exogenous contrast agents, and subregions were segmented based on a k-means cluster model. Subregions of the hippocampus were analyzed (cornu ammonis CA1, CA3, dentate gyrus DG, and ventricle) in order to associate local MRI-observable changes in glutamate with histological measures of glial cell proliferation (GFAP), synapse density (synaptophysin, VGlut1) and glutamate receptor (NMDA-NR1) levels. Early differences in GluCEST between healthy and tauopathy mice were measured in the CA1 and DG subregions (30% reduction, P ≤ 0.001). Synapse density was also significantly reduced in every subregion of the hippocampus in tauopathy mice by 6 months. Volume was not significantly reduced in any subregion until 13 months. Further, a gradient in glutamate levels was observed in vivo along hippocampal axes that became polarized as tauopathy progressed. Dynamics in hippocampal glutamate levels throughout lifetime were most closely correlated with combined changes in synaptophysin and GFAP, indicating that GluCEST imaging may be a surrogate marker of glutamate concentration in glial cells and at the synaptic level. © 2016 Wiley Periodicals, Inc.

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.

High copy wildtype human 1N4R tau expression promotes early pathological tauopathy accompanied by cognitive deficits without progressive neurofibrillary degeneration.

INTRODUCTION: Accumulation of insoluble conformationally altered hyperphosphorylated tau occurs as part of the pathogenic process in Alzheimer's disease (AD) and other tauopathies. In most AD subjects, wild-type (WT) tau aggregates and accumulates in neurofibrillary tangles and dystrophic neurites in the brain; however, in some familial tauopathy disorders, mutations in the gene encoding tau cause disease. RESULTS: We generated a mouse model, Tau4RTg2652, that expresses high levels of normal human tau in neurons resulting in the early stages of tau pathology. In this model, over expression of WT human tau drives pre-tangle pathology in young mice resulting in behavioral deficits. These changes occur at a relatively young age and recapitulate early pre-tangle stages of tau pathology associated with AD and mild cognitive impairment. Several features distinguish the Tau4RTg2652 model of tauopathy from previously described tau transgenic mice. Unlike other mouse models where behavioral and neuropathologic changes are induced by transgenic tau harboring MAPT mutations pathogenic for frontotemporal lobar degeneration (FTLD), the mice described here express the normal tau sequence. CONCLUSIONS: Features of Tau4RTg2652 mice distinguishing them from other established wild type tau overexpressing mice include very early phenotypic manifestations, non-progressive tau pathology, abundant pre-tangle and phosphorylated tau, sparse oligomeric tau species, undetectable fibrillar tau pathology, stability of tau transgene copy number/expression, and normal lifespan. These results suggest that Tau4RTg2652 animals may facilitate studies of tauopathy target engagement where WT tau is driving tauopathy phenotypes.

Differential induction and spread of tau pathology in young PS19 tau transgenic mice following intracerebral injections of pathological tau from Alzheimer's disease or corticobasal degeneration brains.

Filamentous tau pathologies are hallmark lesions of several neurodegenerative tauopathies including Alzheimer's disease (AD) and corticobasal degeneration (CBD) which show cell type-specific and topographically distinct tau inclusions. Growing evidence supports templated transmission of tauopathies through functionally interconnected neuroanatomical pathways suggesting that different self-propagating strains of pathological tau could account for the diverse manifestations of neurodegenerative tauopathies. Here, we describe the rapid and distinct cell type-specific spread of pathological tau following intracerebral injections of CBD or AD brain extracts enriched in pathological tau (designated CBD-Tau and AD-Tau, respectively) in young human mutant P301S tau transgenic (Tg) mice (line PS19) ~6-9 months before they show onset of mutant tau transgene-induced tau pathology. At 1 month post-injection of CBD-Tau, tau inclusions developed predominantly in oligodendrocytes of the fimbria and white matter near the injection sites with infrequent intraneuronal tau aggregates. In contrast, injections of AD-Tau in young PS19 mice induced tau pathology predominantly in neuronal perikarya with little or no oligodendrocyte involvement 1 month post-injection. With longer post-injection survival intervals of up to 6 months, CBD-Tau- and AD-Tau-induced tau pathology spread to different brain regions distant from the injection sites while maintaining the cell type-specific pattern noted above. Finally, CA3 neuron loss was detected 3 months post-injection of AD-Tau but not CBD-Tau. Thus, AD-Tau and CBD-Tau represent specific pathological tau strains that spread differentially and may underlie distinct clinical and pathological features of these two tauopathies. Hence, these strains could become targets to develop disease-modifying therapies for CBD and AD.

In vivo measurement of glutamate loss is associated with synapse loss in a mouse model of tauopathy.

Glutamate is the primary excitatory neurotransmitter in the brain, and is implicated in neurodegenerative diseases such as Alzheimer's disease (AD) and several other tauopathies. The current method for measuring glutamate in vivo is proton magnetic resonance spectroscopy ((1)H MRS), although it has poor spatial resolution and weak sensitivity to glutamate changes. In this study, we sought to measure the effect of tau pathology on glutamate levels throughout the brain of a mouse model of tauopathy using a novel magnetic resonance imaging (MRI) technique. We employed glutamate chemical exchange saturation transfer (GluCEST) imaging, which has been previously validated as a complimentary method for measuring glutamate levels with several important advantages over conventional (1)H MRS. We hypothesized that the regional changes in glutamate levels would correlate with histological measurements of pathology including pathological tau, synapse and neuron loss. Imaging and spectroscopy were carried out on tau transgenic mice with the P301S mutation (PS19, n=9) and their wild-type littermates (WT, n=8), followed by immunohistochemistry of their brain tissue. GluCEST imaging resolution allowed for sub-hippocampal analysis of glutamate. Glutamate was significantly decreased by 29% in the CA sub-region of the PS19 hippocampus, and by 15% in the thalamus, where synapse loss was also measured. Glutamate levels and synapse density remained high in the dentate gyrus sub-region of the hippocampus, where neurogenesis is known to occur. The further development of GluCEST imaging for preclinical applications will be valuable, as therapies are being tested in mouse models of tauopathy.