Doxycycline for transgene control disrupts gut microbiome diversity without compromising acute neuroinflammatory response.

The tetracycline transactivator (tTA) system provides controllable transgene expression through oral administration of the broad-spectrum antibiotic doxycycline. Antibiotic treatment for transgene control in mouse models of disease might have undesirable systemic effects resulting from changes in the gut microbiome. Here we assessed the impact of doxycycline on gut microbiome diversity in a tTA-controlled model of Alzheimer's disease and then examined neuroimmune effects of these microbiome alterations following acute LPS challenge. We show that doxycycline decreased microbiome diversity in both transgenic and wild-type mice and that these changes persisted long after drug withdrawal. Despite the change in microbiome composition, doxycycline treatment had minimal effect on basal transcriptional signatures of inflammation the brain or on the neuroimmune response to LPS challenge. Our findings suggest that central neuroimmune responses may be less affected by doxycycline at doses needed for transgene control than by antibiotic cocktails at doses used for experimental microbiome disruption.

Temporal and spatially controlled APP transgene expression using Cre-dependent alleles.

Although a large number of mouse models have been made to study Alzheimer's disease, only a handful allow experimental control over the location or timing of the protein being used to drive pathology. Other fields have used the Cre and the tamoxifen-inducible CreER driver lines to achieve precise spatial and temporal control over gene deletion and transgene expression, yet these tools have not been widely used in studies of neurodegeneration. Here, we describe two strategies for harnessing the wide range of Cre and CreER driver lines to control expression of disease-associated amyloid precursor protein (APP) in modeling Alzheimer's amyloid pathology. We show that CreER-based spatial and temporal control over APP expression can be achieved with existing lines by combining a Cre driver with a tetracycline-transactivator (tTA)-dependent APP responder using a Cre-to-tTA converter line. We then describe a new mouse line that places APP expression under direct control of Cre recombinase using an intervening lox-stop-lox cassette. Mating this allele with a CreER driver allows both spatial and temporal control over APP expression, and with it, amyloid onset. This article has an associated First Person interview with the first author of the paper.

Combinatorial model of amyloid ß and tau reveals synergy between amyloid deposits and tangle formation.

AIMS: To illuminate the pathological synergy between Aß and tau leading to emergence of neurofibrillary tangles (NFT) in Alzheimer's disease (AD), here, we have performed a comparative neuropathological study utilising three distinctive variants of human tau (WT tau, P301L mutant tau and S320F mutant tau). Previously, in non-transgenic mice, we showed that WT tau or P301L tau does not form NFT while S320F tau can spontaneously aggregate into NFT, allowing us to test the selective vulnerability of these different tau conformations to the presence of Aß plaques. METHODS: We injected recombinant AAV-tau constructs into neonatal APP transgenic TgCRND8 mice or into 3-month-old TgCRND8 mice; both cohorts were aged 3 months post injection. This allowed us to test how different tau variants synergise with soluble forms of Aß (pre-deposit cohort) or with frank Aß deposits (post-deposit cohort). RESULTS: Expression of WT tau did not produce NFT or altered Aß in either cohort. In the pre-deposit cohort, S320F tau induced Aß plaque deposition, neuroinflammation and synaptic abnormalities, suggesting that early tau tangles affect the amyloid cascade. In the post-deposit cohort, contemporaneous expression of S320F tau did not exacerbate amyloid pathology, showing a dichotomy in Aß-tau synergy based on the nature of Aß. P301L tau produced NFT-type inclusions in the post-deposit cohort, but not in the pre-deposit cohort, indicating pathological synergy with pre-existing Aß deposits. CONCLUSIONS: Our data show that different tau mutations representing specific folding variants of tau synergise with Aß to different extents, depending on the presence of cerebral deposits.

Il-10 signaling reduces survival in mouse models of synucleinopathy.

Parkinson's disease (PD) and related synucleinopathies are characterized by chronic neuroinflammation leading to the premise that anti-inflammatory therapies could ameliorate synucleinopathy and associated sequelae. To test this idea, we used recombinant adeno-associated viruses (AAV) to express the anti-inflammatory cytokine, Interleukin (Il)-10, in Line M83 transgenic mice that expresses the PD-associated A53T mutant human α-synuclein (αSyn). Contrary to our expectations, we observed that intraspinal Il-10 expression initiated at birth upregulated microgliosis and led to early death in homozygous M83+/+ mice. We further observed that Il-10 preconditioning led to reduced lifespan in the hemizygous M83+/- mice injected with preformed αSyn aggregates in hindlimb muscles. To determine the mechanistic basis for these adverse effects, we took advantage of the I87A variant Il-10 (vIl-10) that has predominantly immunosuppressive properties. Sustained intraspinal expression of vIl-10 in preformed αSyn-aggregate seeded M83+/- mice resulted in earlier death, accelerated αSyn pathology, pronounced microgliosis, and increased apoptosis compared to control mice. AAV-vIl-10 expression robustly induced p62 and neuronal LC3B accumulation in these mice, indicating that Il-10 signaling mediated preconditioning of the neuraxis can potentially exacerbate αSyn accumulation through autophagy dysfunction in the neurons. Together, our data demonstrate unexpected adverse effects of both Il-10 and its immunosuppressive variant, vIl-10, in a mouse model of PD, highlighting the pleiotropic functions of immune mediators and their complex role in non-cell autonomous signaling in neurodegenerative proteinopathies.

Tau-Mediated Dysregulation of Neuroplasticity and Glial Plasticity.

The inability of individual neurons to compensate for aging-related damage leads to a gradual loss of functional plasticity in the brain accompanied by progressive impairment in learning and memory. Whereas this loss in neuroplasticity is gradual during normal aging, in neurodegenerative diseases such as Alzheimer's disease (AD), this loss is accelerated dramatically, leading to the incapacitation of patients within a decade of onset of cognitive symptoms. The mechanisms that underlie this accelerated loss of neuroplasticity in AD are still not completely understood. While the progressively increasing proteinopathy burden, such as amyloid ß (Aß) plaques and tau tangles, definitely contribute directly to a neuron's functional demise, the role of non-neuronal cells in controlling neuroplasticity is slowly being recognized as another major factor. These non-neuronal cells include astrocytes, microglia, and oligodendrocytes, which through regulating brain homeostasis, structural stability, and trophic support, play a key role in maintaining normal functioning and resilience of the neuronal network. It is believed that chronic signaling from these cells affects the homeostatic network of neuronal and non-neuronal cells to an extent to destabilize this harmonious milieu in neurodegenerative diseases like AD. Here, we will examine the experimental evidence regarding the direct and indirect pathways through which astrocytes and microglia can alter brain plasticity in AD, specifically as they relate to the development and progression of tauopathy. In this review article, we describe the concepts of neuroplasticity and glial plasticity in healthy aging, delineate possible mechanisms underlying tau-induced plasticity dysfunction, and discuss current clinical trials as well as future disease-modifying approaches.

Intracerebral Expression of AAV-APOE4 Is Not Sufficient to Alter Tau Burden in Two Distinct Models of Tauopathy.

Apolipoprotein E4 (APOE4) is the major genetic risk factor for sporadic Alzheimer's disease (AD), which is characterized by amyloid ß (Aß) plaques and tau tangles. Though the role of APOE4 in Aß pathogenesis has been mechanistically defined in rodent models, much less is known regarding the relationship of APOE4 to tau pathogenesis. Recent studies have indicated a possible correlation between APOE isoform-dependent alterations in tau pathology and neurodegeneration. To explore whether neuronal expression of APOE4 triggers tauopathy, here we delivered adeno-associated viruses (AAV) expressing human APOE4 in two different models of tauopathy-rTg4510 and PS19 lines. Intracerebroventricular delivery of AAV-APOE4 in neonatal rTg4510 and PS19 mice resulted in increased APOE4 protein in neurons but did not result in altered phosphorylated tau burden, pretangle tau pathology, or silver-positive tangle pathology. Biochemical analysis of synaptic proteins did not reveal substantial alterations. Our results indicate that over-expression of APOE4 in neurons, using an AAV-mediated approache, is not sufficient to accelerate or otherwise alter the inherent tau pathology that occurs in mice overexpressing mutant human tau.

Combining P301L and S320F tau variants produces a novel accelerated model of tauopathy.

Understanding the biological functions of tau variants can illuminate differential etiologies of Alzheimer's disease (AD) and primary tauopathies. Though the end-stage neuropathological attributes of AD and primary tauopathies are similar, the etiology and behavioral outcomes of these diseases follow unique and divergent trajectories. To study the divergent physiological properties of tau variants on a uniform immunogenetic background, we created somatic transgenesis CNS models of tauopathy utilizing neonatal delivery of adeno-associated viruses expressing wild-type (WT) or mutant tau in non-transgenic mice. We selected four different tau variants-WT tau associated with AD, P301L mutant tau associated with frontotemporal dementia (FTD), S320F mutant tau associated with Pick's disease and a combinatorial approach using P301L/S320F mutant tau. CNS-targeted expression of WT and P301L mutant tau results in robust tau hyperphosphorylation without tangle pathology, gradually developing age-progressive memory deficits. In contrast, the S320F variant, especially in combination with P301L, produces an AD-type tangle pathology, focal neuroinflammation and memory impairment on an accelerated time scale. Using the doubly mutated P301L/S320F tau variant, we demonstrate that combining different mutations can have an additive effect on neuropathologies and associated co-morbidities, possibly hinting at involvement of unique functional pathways. Importantly, we also show that overexpression of wild-type tau as well as an FTD-associated tau variant can lead to cognitive deficits even in the absence of tangles. Together, our data highlights the synergistic neuropathologies and associated cognitive and synaptic alterations of the combinatorial tau variant leading to a robust model of tauopathy.

Genetic Deletion of PGF2α-FP Receptor Exacerbates Brain Injury Following Experimental Intracerebral Hemorrhage.

Background: The release of inflammatory molecules such as prostaglandins (e.g., PGF2α) is associated with brain damage following an intracerebral hemorrhagic (ICH) stroke; however, the role of PGF2α and its cognate FP receptor in ICH remains unclear. This study focused on investigating the role of the FP receptor as a target for novel neuroprotective drugs in a preclinical model of ICH, aiming to investigate the contribution of the PGF2α-FP axis in modulating functional recovery and anatomical outcomes following ICH. Results: Neurological deficit scores in FP-/- mice were significantly higher compared to WT mice 72 h after ICH (6.1 ± 0.7 vs. 3.1 ± 0.8; P < 0.05). Assessing motor skills, the total time mice stayed on the rotating rod was significantly less in FP-/-mice compared to WT mice 24 h after ICH (27.0 ± 7.5 vs. 52.4 ± 11.2 s; P < 0.05). Using grip strength to quantify forepaw strength, results showed that the FP-/- mice had significantly less strength compared to WT mice 72 h after ICH (96.4 ± 17.0 vs. 129.6 ± 5.9 g; P < 0.01). In addition to the behavioral outcomes, histopathological measurements were made. In Cresyl violet stained brain sections, the FP-/- mice showed a significantly larger lesion volume compared to the WT (15.0 ± 2.2 vs. 3.2 ± 1.7 mm3; P < 0.05 mice.) To estimate the presence of ferric iron in the peri-hematoma area, Perls' staining was performed, which revealed that FP-/- mice had significantly greater staining than the WT mice (186.3 ± 34.4% vs. 86.9 ± 13.0% total positive pixel counts, P < 0.05). Immunoreactivity experiments on brain sections from FP-/- and WT mice post-ICH were performed to monitor changes in microgliosis and astrogliosis using antibodies against Iba1 and GFAP respectively. These experiments showed that FP-/- mice had a trend toward greater astrogliosis than WT mice post-ICH. Conclusion: We showed that deletion of the PGF2α FP receptor exacerbates behavioral impairments and increases lesion volumes following ICH compared to WT-matched controls.Detailed mechanisms responsible for these novel results are actively being pursued.

TLR5 decoy receptor as a novel anti-amyloid therapeutic for Alzheimer's disease.

There is considerable interest in harnessing innate immunity to treat Alzheimer's disease (AD). Here, we explore whether a decoy receptor strategy using the ectodomain of select TLRs has therapeutic potential in AD. AAV-mediated expression of human TLR5 ectodomain (sTLR5) alone or fused to human IgG4 Fc (sTLR5Fc) results in robust attenuation of amyloid ß (Aß) accumulation in a mouse model of Alzheimer-type Aß pathology. sTLR5Fc binds to oligomeric and fibrillar Aß with high affinity, forms complexes with Aß, and blocks Aß toxicity. Oligomeric and fibrillar Aß modulates flagellin-mediated activation of human TLR5 but does not, by itself, activate TLR5 signaling. Genetic analysis shows that rare protein coding variants in human TLR5 may be associated with a reduced risk of AD. Further, transcriptome analysis shows altered TLR gene expression in human AD. Collectively, our data suggest that TLR5 decoy receptor-based biologics represent a novel and safe Aß-selective class of biotherapy in AD.

Ifngr1 and Stat1 mediated canonical Ifn-γ signaling drives nigrostriatal degeneration.

Brain expression of AAV-Ifn-γ leads to reactive gliosis, nigrostriatal degeneration and midbrain calcification in wild type mice. This mouse model phenocopies idiopathic basal ganglia calcification which is associated with Parkinsonian symptoms. To understand how the nigro-striatal pathway is selectively vulnerable to Ifn-γ, we determined if the phenotype is driven by canonical signaling intermediates, Ifngr1 and Stat1. Using focused bioinformatic analysis and rotarod testing, we show that neuroinflammation and motor abnormalities precede the appearance of midbrain neuropathologies in the brains of Ifn-γ mouse model. To test whether canonical Ifn-γ signaling is a key driver of progressive nigrostriatal degeneration, we overexpressed Ifn-γ in the brains of Ifngr1-/- and Stat1-/- mice. Expression of Ifn-γ in Ifngr1-/- mice did not result in any neuroinflammation, midbrain calcinosis or nigrostriatal degenerative pathology. Interestingly, in Stat1-/- mice, Ifn-γ expression resulted in gliosis without recapitulating the neurodegenerative phenotype. Overall, our data shows that canonical Ifn-γ signaling triggers midbrain calcinosis and nigrostriatal neurodegeneration, providing mechanistic insights into cytokine-driven selective neuronal vulnerability. Our study establishes the broader relevance of inflammatory signaling in neurodegenerative diseases and can potentially identify novel immunological targets for Parkinsonian syndromes.

Inflammatory pre-conditioning restricts the seeded induction of α-synuclein pathology in wild type mice.

BACKGROUND: Cell-to-cell transmission of α-synuclein (αSyn) is hypothesized to play an important role in disease progression in synucleinopathies. This process involves cellular uptake of extracellular amyloidogenic αSyn seeds followed by seeding of endogenous αSyn. Though it is well known that αSyn is an immunogenic protein that can interact with immune receptors, the role of innate immunity in regulating induction of αSyn pathology in vivo is unknown. Herein, we explored whether altering innate immune activation affects induction of αSyn pathology in wild type mice. METHODS: We have previously demonstrated that recombinant adeno-associated virus (AAV) mediated expression of the inflammatory cytokine, Interleukin (IL)-6, in neonatal wild type mice brains leads to widespread immune activation in the brain without overt neurodegeneration. To investigate how IL-6 expression affects induction of αSyn pathology, we injected mouse wild type αSyn fibrils in the hippocampus of AAV-IL-6 expressing mice. Control mice received AAV containing an Empty vector (EV) construct. Two separate cohorts of AAV-IL-6 and AAV-EV mice were analyzed in this study: 4 months or 2 months following intrahippocampal αSyn seeding. RESULTS: Here, we show that IL-6 expression resulted in widespread gliosis and concurrently reduced αSyn inclusion pathology induced by a single intra-hippocampal injection of exogenous amyloidogenic αSyn. The reduction in αSyn inclusion pathology in IL-6 expressing mice was time-dependent. Suppression of αSyn pathology was accompanied by reductions in both argyrophilic and p62 immunoreactive inclusions. CONCLUSIONS: Our data supports a beneficial role of inflammatory priming of the CNS in wild type mice challenged with exogenous αSyn. A likely mechanism is efficient astroglial scavenging of exogenous αSyn, at least early in the disease process, and in the absence of human αSyn transgene overexpression. Given evidence that a pro-inflammatory environment may restrict seeding of αSyn pathology, this can be used to design anti-αSyn immunobiotherapies by harnessing innate immune function.