Persistence and Sexual Dimorphism of Gut Dysbiosis and Pathobiome after Sepsis and Trauma.

OBJECTIVE: To evaluate the persistence of intestinal microbiome dysbiosis and gut-plasma metabolomic perturbations following severe trauma or sepsis weeks after admission in patients experiencing chronic critical illness (CCI). SUMMARY: Trauma and sepsis can lead to gut dysbiosis and alterations in the plasma and fecal metabolome. However, the impact of these perturbations and correlations between gut dysbiosis and the plasma metabolome in chronic critical illness have not been studied. METHODS: A prospective observational cohort study was performed with healthy subjects, severe trauma patients, patients with sepsis residing in an intensive care unit (ICU) for 2-3 weeks. A high-throughput multi-omics approach was utilized to evaluate the gut microbial and gut-plasma metabolite responses in critically ill trauma and sepsis patients 14-21 days after ICU admission. RESULTS: Patients in the sepsis and trauma cohorts demonstrated strikingly depleted gut microbiome diversity, with significant alterations and specific pathobiome patterns in the microbiota composition compared to healthy subjects. Further subgroup analyses based on sex revealed resistance to changes in microbiome diversity among female trauma patients compared to healthy counterparts. Sex-specific changes in fecal metabolites were also observed after trauma and sepsis, while plasma metabolite changes were similar in both males and females. CONCLUSIONS: Dysbiosis induced by trauma and sepsis persists up to 14-21 days after onset and is sex-specific, underscoring the implication of pathobiome and entero-septic microbial-metabolite perturbations in post-sepsis and post-trauma CCI. This indicates resilience to infection or injury in females' microbiome and should inform and facilitate future precision/personalized medicine strategies in the intensive care unit.

Deletion of Abi3/Gngt2 influences age-progressive amyloid ß and tau pathologies in distinctive ways.

BACKGROUND: The S209F variant of Abelson Interactor Protein 3 (ABI3) increases risk for Alzheimer's disease (AD), but little is known about its function in relation to AD pathogenesis. METHODS: Here, we use a mouse model that is deficient in Abi3 locus to study how the loss of function of Abi3 impacts two cardinal neuropathological hallmarks of AD-amyloid ß plaques and tau pathology. Our study employs extensive neuropathological and transcriptomic characterization using transgenic mouse models and adeno-associated virus-mediated gene targeting strategies. RESULTS: Analysis of bulk RNAseq data confirmed age-progressive increase in Abi3 levels in rodent models of AD-type amyloidosis and upregulation in AD patients relative to healthy controls. Using RNAscope in situ hybridization, we localized the cellular distribution of Abi3 in mouse and human brains, finding that Abi3 is expressed in both microglial and non-microglial cells. Next, we evaluated Abi3-/- mice and document that both Abi3 and its overlapping gene, Gngt2, are disrupted in these mice. Using multiple transcriptomic datasets, we show that expression of Abi3 and Gngt2 are tightly correlated in rodent models of AD and human brains, suggesting a tight co-expression relationship. RNAseq of the Abi3-Gngt2-/- mice revealed upregulation of Trem2, Plcg2, and Tyrobp, concomitant with induction of an AD-associated neurodegenerative signature, even in the absence of AD-typical neuropathology. In APP mice, loss of Abi3-Gngt2 resulted in a gene dose- and age-dependent reduction in Aß deposition. Additionally, in Abi3-Gngt2-/- mice, expression of a pro-aggregant form of human tau exacerbated tauopathy and astrocytosis. Further, using in vitro culture assays, we show that the AD-associated S209F mutation alters the extent of ABI3 phosphorylation. CONCLUSIONS: These data provide an important experimental framework for understanding the role of Abi3-Gngt2 function and early inflammatory gliosis in AD. Our studies also demonstrate that inflammatory gliosis could have opposing effects on amyloid and tau pathology, highlighting the unpredictability of targeting immune pathways in AD.

Modulating innate immune activation states impacts the efficacy of specific Aß immunotherapy.

INTRODUCTION: Passive immunotherapies targeting Aß continue to be evaluated as Alzheimer's disease (AD) therapeutics, but there remains debate over the mechanisms by which these immunotherapies work. Besides the amount of preexisting Aß deposition and the type of deposit (compact or diffuse), there is little data concerning what factors, independent of those intrinsic to the antibody, might influence efficacy. Here we (i) explored how constitutive priming of the underlying innate activation states by Il10 and Il6 might influence passive Aß immunotherapy and (ii) evaluated transcriptomic data generated in the AMP-AD initiative to inform how these two cytokines and their receptors' mRNA levels are altered in human AD and an APP mouse model. METHODS: rAAV2/1 encoding EGFP, Il6 or Il10 were delivered by somatic brain transgenesis to neonatal (P0) TgCRND8 APP mice. Then, at 2 months of age, the mice were treated bi-weekly with a high-affinity anti-Aß1-16 mAb5 monoclonal antibody or control mouse IgG until 6 months of age. rAAV mediated transgene expression, amyloid accumulation, Aß levels and gliosis were assessed. Extensive transcriptomic data was used to evaluate the mRNA expression levels of IL10 and IL6 and their receptors in the postmortem human AD temporal cortex and in the brains of TgCRND8 mice, the later at multiple ages. RESULTS: Priming TgCRND8 mice with Il10 increases Aß loads and blocks efficacy of subsequent mAb5 passive immunotherapy, whereas priming with Il6 priming reduces Aß loads by itself and subsequent Aß immunotherapy shows only a slightly additive effect. Transcriptomic data shows that (i) there are significant increases in the mRNA levels of Il6 and Il10 receptors in the TgCRND8 mouse model and temporal cortex of humans with AD and (ii) there is a great deal of variance in individual mouse brain and the human temporal cortex of these interleukins and their receptors. CONCLUSIONS: The underlying immune activation state can markedly affect the efficacy of passive Aß immunotherapy. These results have important implications for ongoing human AD immunotherapy trials, as they indicate that underlying immune activation states within the brain, which may be highly variable, may influence the ability for passive immunotherapy to alter Aß deposition.

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.

A novel tau-based rhesus monkey model of Alzheimer's pathogenesis.

INTRODUCTION: Alzheimer's disease (AD) is a devastating condition with no effective treatments, with promising findings in rodents failing to translate into successful therapies for patients. METHODS: Targeting the vulnerable entorhinal cortex (ERC), rhesus monkeys received two injections of an adeno-associated virus expressing a double tau mutation (AAV-P301L/S320F) in the left hemisphere, and control AAV-green fluorescent protein in the right ERC. Noninjected aged-matched monkeys served as additional controls. RESULTS: Within 3 months we observed evidence of misfolded tau propagation, similar to what is hypothesized to occur in humans. Viral delivery of human 4R-tau also coaptates monkey 3R-tau via permissive templating. Tau spreading is accompanied by robust neuroinflammatory response driven by TREM2+ microglia, with biomarkers of inflammation and neuronal loss in the cerebrospinal fluid and plasma. DISCUSSION: These results highlight the initial stages of tau seeding and propagation in a primate model, a more powerful translational approach for the development of new therapies for AD.

Integrative functional genomic analysis of intron retention in human and mouse brain with Alzheimer's disease.

Intron retention (IR) has been implicated in the pathogenesis of complex diseases such as cancers; its association with Alzheimer's disease (AD) remains unexplored. We performed genome-wide analysis of IR through integrating genetic, transcriptomic, and proteomic data of AD subjects and mouse models from the Accelerating Medicines Partnership-Alzheimer's Disease project. We identified 4535 and 4086 IR events in 2173 human and 1736 mouse genes, respectively. Quantitation of IR enabled the identification of differentially expressed genes that conventional exon-level approaches did not reveal. There were significant correlations of intron expression within innate immune genes, like HMBOX1, with AD in humans. Peptides with a high probability of translation from intron-retained mRNAs were identified using mass spectrometry. Further, we established AD-specific intron expression Quantitative Trait Loci, and identified splicing-related genes that may regulate IR. Our analysis provides a novel resource for the search for new AD biomarkers and pathological mechanisms.

IL-10 based immunomodulation initiated at birth extends lifespan in a familial mouse model of amyotrophic lateral sclerosis.

Inflammatory signaling is thought to modulate the neurodegenerative cascade in amyotrophic lateral sclerosis (ALS). We have previously shown that expression of Interleukin-10 (IL-10), a classical anti-inflammatory cytokine, extends lifespan in the SOD1-G93A mouse model of familial ALS. Here we test whether co-expression of the decoy chemokine receptor M3, that can scavenge inflammatory chemokines, augments the efficacy of IL-10. We found that recombinant adeno-associated virus (AAV)-mediated expression of IL-10, alone, or in combination with M3, resulted in modest extension of lifespan relative to control SOD1-G93A cohort. Interestingly neither AAV-M3 alone nor AAV-IL-10 + AAV-M3 extend survival beyond that of the AAV-IL-10 alone cohort. Focused transcriptomic analysis revealed induction of innate immunity and phagocytotic pathways in presymptomatic SOD1-G93A mice expressing IL-10 + M3 or IL-10 alone. Further, while IL-10 expression increased microglial burden, the IL-10 + M3 group showed lower microglial burden, suggesting that M3 can successfully lower microgliosis before disease onset. Our data demonstrates that over-expression of an anti-inflammatory cytokine and a decoy chemokine receptor can modulate inflammatory processes in SOD1-G93A mice, modestly delaying the age to paralysis. This suggests that multiple inflammatory pathways can be targeted simultaneously in neurodegenerative disease and supports consideration of adapting these approaches to treatment of ALS and related disorders.

Diversity in Aß deposit morphology and secondary proteome insolubility across models of Alzheimer-type amyloidosis.

A hallmark pathology of Alzheimer's disease (AD) is the formation of amyloid ß (Aß) deposits that exhibit diverse localization and morphologies, ranging from diffuse to cored-neuritic deposits in brain parenchyma, with cerebral vascular deposition in leptomeningeal and parenchymal compartments. Most AD brains exhibit the full spectrum of pathologic Aß morphologies. In the course of studies to model AD amyloidosis, we have generated multiple transgenic mouse models that vary in the nature of the transgene constructs that are expressed; including the species origin of Aß peptides, the levels and length of Aß that is deposited, and whether mutant presenilin 1 (PS1) is co-expressed. These models recapitulate features of human AD amyloidosis, but interestingly some models can produce pathology in which one type of Aß morphology dominates. In prior studies of mice that primarily develop cored-neuritic deposits, we determined that Aß deposition is associated with changes in cytosolic protein solubility in which a subset of proteins become detergent-insoluble, indicative of secondary proteome instability. Here, we survey changes in cytosolic protein solubility across seven different transgenic mouse models that exhibit a range of Aß deposit morphologies. We find a surprisingly diverse range of changes in proteome solubility across these models. Mice that deposit human Aß40 and Aß42 in cored-neuritic plaques had the most robust changes in proteome solubility. Insoluble cytosolic proteins were also detected in the brains of mice that develop diffuse Aß42 deposits but to a lesser extent. Notably, mice with cored deposits containing only Aß42 had relatively few proteins that became detergent-insoluble. Our data provide new insight into the diversity of biological effects that can be attributed to different types of Aß pathology and support the view that fibrillar cored-neuritic plaque pathology is the more disruptive Aß pathology in the Alzheimer's cascade.

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.

Diffusion magnetic resonance imaging-derived free water detects neurodegenerative pattern induced by interferon-γ.

Imaging biomarkers for immune activation may be valuable for early-stage detection, therapeutic testing, and research on neurodegenerative conditions. In the present study, we determined whether diffusion magnetic resonance imaging-derived free water signal is a sensitive marker for neuroinflammatory effects of interferon-gamma (Ifn-γ). Neonatal wild-type mice were injected in the cerebral ventricles with recombinant adeno-associated viruses expressing the inflammatory cytokine Ifn-γ. Groups of mice expressing Ifn-γ and age-matched controls were imaged at 1, 5 and 8 months. Mice deficient in Ifngr1-/- and Stat1-/- were scanned at 5 months as controls for the signaling cascades activated by Ifn-γ. The results indicate that Ifn-γ affected fractional anisotropy (FA), mean diffusivity (MD), and free water (FW) in white matter structures, midline cortical areas, and medial thalamic areas. In these structures, FA and MD decreased progressively from 1 to 8 months of age, while FW increased significantly. The observed reductions in FA and MD and increased FW with elevated brain Ifn-γ was not observed in Ifngr1-/- or Stat1-/- mice. These results suggest that the observed microstructure changes involve the Ifn-gr1 and Stat1 signaling. Interestingly, increases in FW were observed in midbrain of Ifngr1-/- mice, which suggests alternative Ifn-γ signaling in midbrain. Although initial evidence is offered in relation to the sensitivity of the FW signal to neurodegenerative and/or inflammatory patterns specific to Ifn-γ, further research is needed to determine applicability and specificity across animal models of neuroinflammatory and degenerative disorders.

Neurite orientation dispersion and density imaging reveals white matter and hippocampal microstructure changes produced by Interleukin-6 in the TgCRND8 mouse model of amyloidosis.

Extracellular ß-amyloid (Aß) plaque deposits and inflammatory immune activation are thought to alter various aspects of tissue microstructure, such as extracellular free water, fractional anisotropy and diffusivity, as well as the density and geometric arrangement of axonal processes. Quantifying these microstructural changes in Alzheimer's disease and related neurodegenerative dementias could serve to monitor or predict disease course. In the present study we used high-field diffusion magnetic resonance imaging (dMRI) to investigate the effects of Aß and inflammatory interleukin-6 (IL6), alone or in combination, on in vivo tissue microstructure in the TgCRND8 mouse model of Alzheimer's-type Aß deposition. TgCRND8 and non-transgenic (nTg) mice expressing brain-targeted IL6 or enhanced glial fibrillary protein (EGFP controls) were scanned at 8 months of age using a 2-shell, 54-gradient direction dMRI sequence at 11.1 T. Images were processed using the diffusion tensor imaging (DTI) model or the neurite orientation dispersion and density imaging (NODDI) model. DTI and NODDI processing in TgCRND8 mice revealed a microstructure pattern in white matter (WM) and hippocampus consistent with radial and longitudinal diffusivity deficits along with an increase in density and geometric complexity of axonal and dendritic processes. This included reduced FA, mean, axial and radial diffusivity, and increased orientation dispersion (ODI) and intracellular volume fraction (ICVF) measured in WM and hippocampus. IL6 produced a 'protective-like' effect on WM FA in TgCRND8 mice, observed as an increased FA that counteracted a reduction in FA observed with endogenous Aß production and accumulation. In addition, we found that ICVF and ODI had an inverse relationship with the functional connectome clustering coefficient. The relationship between NODDI and graph theory metrics suggests that currently unknown microstructure alterations in WM and hippocampus are associated with diminished functional network organization in the brain.

An anti-CRF antibody suppresses the HPA axis and reverses stress-induced phenotypes.

Hypothalamic-pituitary-adrenal (HPA) axis dysfunction contributes to numerous human diseases and disorders. We developed a high-affinity monoclonal antibody, CTRND05, targeting corticotropin-releasing factor (CRF). In mice, CTRND05 blocks stress-induced corticosterone increases, counteracts effects of chronic variable stress, and induces other phenotypes consistent with suppression of the HPA axis. CTRND05 induces skeletal muscle hypertrophy and increases lean body mass, effects not previously reported with small-molecule HPA-targeting pharmacologic agents. Multiorgan transcriptomics demonstrates broad HPA axis target engagement through altering levels of known HPA-responsive transcripts such as Fkbp5 and Myostatin and reveals novel HPA-responsive pathways such as the Apelin-Apelin receptor system. These studies demonstrate the therapeutic potential of CTRND05 as a suppressor of the HPA axis and serve as an exemplar of a potentially broader approach to target neuropeptides with immunotherapies, as both pharmacologic tools and novel therapeutics.

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.

APP-Mediated Signaling Prevents Memory Decline in Alzheimer's Disease Mouse Model.

Amyloid precursor protein (APP) and its metabolites play key roles in Alzheimer's disease (AD) pathophysiology. Whereas short amyloid-ß (Aß) peptides derived from APP are pathogenic, the APP holoprotein serves multiple purposes in the nervous system through its cell adhesion and receptor-like properties. Our studies focused on the signaling mediated by the APP cytoplasmic tail. We investigated whether sustained APP signaling during brain development might favor neuronal plasticity and memory process through a direct interaction with the heterotrimeric G-protein subunit GαS (stimulatory G-protein alpha subunit). Our results reveal that APP possesses autonomous regulatory capacity within its intracellular domain that promotes APP cell surface residence, precludes Aß production, facilitates axodendritic development, and preserves cellular substrates of memory. Altogether, these events contribute to strengthening cognitive functions and are sufficient to modify the course of AD pathology.

rAAV-based brain slice culture models of Alzheimer's and Parkinson's disease inclusion pathologies.

It has been challenging to produce ex vivo models of the inclusion pathologies that are hallmark pathologies of many neurodegenerative diseases. Using three-dimensional mouse brain slice cultures (BSCs), we have developed a paradigm that rapidly and robustly recapitulates mature neurofibrillary inclusion and Lewy body formation found in Alzheimer's and Parkinson's disease, respectively. This was achieved by transducing the BSCs with recombinant adeno-associated viruses (rAAVs) that express α-synuclein or variants of tau. Notably, the tauopathy BSC model enables screening of small molecule therapeutics and tracking of neurodegeneration. More generally, the rAAV BSC "toolkit" enables efficient transduction and transgene expression from neurons, microglia, astrocytes, and oligodendrocytes, alone or in combination, with transgene expression lasting for many months. These rAAV-based BSC models provide a cost-effective and facile alternative to in vivo studies, and in the future can become a widely adopted methodology to explore physiological and pathological mechanisms related to brain function and dysfunction.

Alzheimer's disease phospholipase C-gamma-2 (PLCG2) protective variant is a functional hypermorph.

BACKGROUND: Recent Genome Wide Association Studies (GWAS) have identified novel rare coding variants in immune genes associated with late onset Alzheimer's disease (LOAD). Amongst these, a polymorphism in phospholipase C-gamma 2 (PLCG2) P522R has been reported to be protective against LOAD. PLC enzymes are key elements in signal transmission networks and are potentially druggable targets. PLCG2 is highly expressed in the hematopoietic system. Hypermorphic mutations in PLCG2 in humans have been reported to cause autoinflammation and immune disorders, suggesting a key role for this enzyme in the regulation of immune cell function. METHODS: We assessed PLCG2 distribution in human and mouse brain tissue via immunohistochemistry and in situ hybridization. We transfected heterologous cell systems (COS7 and HEK293T cells) to determine the effect of the P522R AD-associated variant on enzymatic function using various orthogonal assays, including a radioactive assay, IP-One ELISA, and calcium assays. RESULTS: PLCG2 expression is restricted primarily to microglia and granule cells of the dentate gyrus. Plcg2 mRNA is maintained in plaque-associated microglia in the cerebral tissue of an AD mouse model. Functional analysis of the p.P522R variant demonstrated a small hypermorphic effect of the mutation on enzyme function. CONCLUSIONS: The PLCG2 P522R variant is protective against AD. We show that PLCG2 is expressed in brain microglia, and the p.P522R polymorphism weakly increases enzyme function. These data suggest that activation of PLCγ2 and not inhibition could be therapeutically beneficial in AD. PLCγ2 is therefore a potential target for modulating microglia function in AD, and a small molecule drug that weakly activates PLCγ2 may be one potential therapeutic approach.

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.

Combination of Aß Suppression and Innate Immune Activation in the Brain Significantly Attenuates Amyloid Plaque Deposition.

Anti-Aß clinical trials are currently under way to determine whether preventing amyloid deposition will be beneficial in arresting progression of Alzheimer disease. Both clinical and preclinical studies suggest that antiamyloid strategies are only effective if started at early stages of the disease process in a primary prevention strategy. Because this approach will be difficult to deploy, strategies for secondary prevention aimed at later stages of disease are also needed. In this study, we asked whether combining innate immune activation in the brain with concurrent Aß suppression could enhance plaque clearance and could improve pathologic outcomes in mice with moderate amyloid pathologic disorder. Starting at 5 months of age, tet-off amyloid precursor protein transgenic mice were treated with doxycycline (dox) to suppress further amyloid precursor protein/Aß production, and at the same time mice were intracranially injected with adeno-associated virus 1 expressing murine IL-6 (AAV1-mIL-6). Three months later, mice treated with the combination of Aß suppression and AAV1-mIL-6 showed significantly less plaque pathologic disorder than dox or AAV1-mIL-6 only groups. The combination of AAV1-mIL-6 + dox treatment lowered total plaque burden by >60% versus untreated controls. Treatment with either dox or AAV1-mIL-6 alone was less effective than the combination. Our results suggest a synergistic mechanism by which the up-regulation of mIL-6 was able to improve plaque clearance in the setting of Aß suppression.

Generation and characterization of new monoclonal antibodies targeting the PHF1 and AT8 epitopes on human tau.

Tauopathies are a group of neurodegenerative disorders, including Alzheimer's disease, defined by the presence of brain pathological inclusions comprised of abnormally aggregated and highly phosphorylated tau protein. The abundance of brain tau aggregates correlates with disease severity and select phospho-tau epitopes increase at early stages of disease. We generated and characterized a series of novel monoclonal antibodies directed to tau phosphorylated at several of these phospho-epitopes, including Ser396/Ser404, Ser404 and Thr205. We also generated phosphorylation independent antibodies against amino acid residues 193-211. We show that most of these antibodies are highly specific for tau and strongly recognize pathological inclusions in human brains and in a transgenic mouse model of tauopathy. They also reveal epitope-specific differences in the biochemical properties of Alzheimer's disease sarkosyl-insoluble tau. These new reagents will be useful for investigating the progression of tau pathology and further as tools to target the cellular transmission of tau pathology.

Amyloid ß peptides overexpression in retinal pigment epithelial cells via AAV-mediated gene transfer mimics AMD-like pathology in mice.

Age-related macular degeneration (AMD) is a progressive retinal neurodegenerative disorder characterized by extracellular deposits known as drusen. A major constituent of drusen deposits are Alzheimer disease-associated amyloid ß (Aß) peptides. To understand the etiology of Aß proteostasis in AMD, we delivered recombinant adeno-associated virus (AAV) encoding Aß42 and Aß40 peptides fused to BRI2 protein by intraocular injection in C57BL/6J mice. Endogenous protease cleavage of such constructs leads to production of secreted Aß42 and Aß40 respectively. We demonstrate that overexpression of secreted Aß40 or Aß42 resulted in dramatic induction of drusen-like deposits by 2 months' post-injection. These drusen-like deposits were immunopositive for Aß and complement proteins but did not stain for conventional amyloid dyes, such as Thioflavin S. Both injected cohorts showed gliosis and degenerative changes, though ERG responses were minimally affected. Intriguingly, simultaneous overexpression of BRI-Aß40 or BRI-Aß42 together resulted in dose-dependent and cumulative changes reminiscent of AMD type pathology - drusen-like deposits, severe reduction in ERG responses, photoreceptor cell loss and gliosis. Here, we have established a physiological model of Aß containing deposits in wild-type mice that recapitulates major retinal pathophysiological features of AMD and will be instrumental in mechanistic understanding and development of therapeutic strategies against AMD.