Intracranial injection of AAV expressing NEP but not IDE reduces amyloid pathology in APP+PS1 transgenic mice

The accumulation of β-amyloid peptides in the brain has been recognized as an essential factor in Alzheimer\u27s disease pathology. Several proteases, including Neprilysin (NEP), endothelin converting enzyme (ECE), and insulin degrading enzyme (IDE), have been shown to cleave β-amyloid peptides (Aβ). We have previously reported reductions in amyloid in APP+PS1 mice with increased expression of ECE. In this study we compared the vector-induced increased expression of NEP and IDE. We used recombinant adeno-associated viral vectors expressing either native forms of NEP (NEP-n) or IDE (IDE-n), or engineered secreted forms of NEP (NEP-s) or IDE (IDE-s). In a six-week study, immunohistochemistry staining for total Aβ was significantly decreased in animals receiving the NEP-n and NEP-s but not for IDE-n or IDE-s in either the hippocampus or cortex. Congo red staining followed a similar trend revealing significant decreases in the hippocampus and the cortex for NEP-n and NEP-s treatment groups. Our results indicate that while rAAV-IDE does not have the same therapeutic potential as rAAV-NEP, rAAV-NEP-s and NEP-n are effective at reducing amyloid loads, and both of these vectors continue to have significant effects nine months post-injection. As such, they may be considered reasonable candidates for gene therapy trials in AD

Hypusination of Eif5a Regulates Cytoplasmic TDP-43 Aggregation and Accumulation in a Stress-Induced Cellular Model

TAR DNA-binding protein 43 (TDP-43) is a nuclear RNA/DNA binding protein involved in mRNA metabolism. Aberrant mislocalization to the cytoplasm and formation of phosphorylated/aggregated TDP-43 inclusions remains the hallmark pathology in a spectrum of neurodegenerative diseases, including frontotemporal disorders and Alzheimer\u27s disease. Eukaryotic Translation Initiation Factor 5A undergoes a unique post-translation modification of lysine to hypusine (K50), which determines eIF5A binding partners. We used a sodium arsenite-induced cellular stress model to investigate the role of hypusinated eIF5A (eIF5AHypK50) in governing TDP-43 cytoplasmic mislocalization and accumulation in stress granule. Our proteomics and functional data provide evidence that eIF5A interacts with TDP-43 in a hypusine-dependent manner. Additionally, we showed that following stress TDP-43 interactions with eIF5AHypK50 were induced both in the cytoplasm and stress granules. Pharmacological reduction of hypusination or mutations of lysine residues within the hypusine loop decreased phosphorylated and insoluble TDP-43 levels. The proteomic and biochemical analysis also identified nuclear pore complex importins KPNA1/2, KPNB1, and RanGTP as interacting partners of eIF5AHypK50. These findings are the first to provide a novel pathway and potential therapeutic targets that require further investigation in models of TDP-43 proteinopathies

LPS- induced inflammation exacerbates phospho-tau pathology in rTg4510 mice

Inflammation and microglial activation are associated with Alzheimer's disease (AD) pathology. Somewhat surprisingly, injection of a prototypical inflammatory agent, lipopolysaccharide (LPS) into brains of amyloid precursor protein (APP) transgenic mice clears some of the pre-existing amyloid deposits. It is less well understood how brain inflammation modulates tau pathology in the absence of Aβ. These studies examined the role of LPS-induced inflammation on tau pathology. We used transgenic rTg4510 mice, which express the P301L mutation (4R0N TauP301L) and initiate tau pathology between 3-5 months of age. First, we found an age-dependent increase in several markers of microglial activation as these rTg4510 mice aged and tau tangles accumulated. LPS injections into the frontal cortex and hippocampus induced significant activation of CD45 and arginase 1 in rTg4510 and non-transgenic mice. In addition, activation of YM1 by LPS was exaggerated in transgenic mice relative to non-transgenic animals. Expression of Ser199/202 and phospho-tau Ser396 was increased in rTg4510 mice that received LPS compared to vehicle injections. However, the numbers of silver-positive neurons, implying presence of more pre- and mature tangles, was not significantly affected by LPS administration. These data suggest that inflammatory stimuli can facilitate tau phosphorylation. Coupled with prior results demonstrating clearance of Aβ by similar LPS injections, these results suggest that brain inflammation may have opposing effects on amyloid and tau pathology, possibly explaining the failures (to date) of anti-inflammatory therapies in AD patients

TDP-43 Mediated Blood-Brain Barrier Permeability and Leukocyte Infiltration Promote Neurodegeneration in a Low-Grade Systemic Inflammation Mouse Model

BACKGROUND: Neuronal cytoplasmic inclusions containing TAR DNA-binding protein 43 (TDP-43) are a neuropathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer\u27s Disease (AD). Emerging evidence also indicates that systemic inflammation may be a contributor to the pathology progression of these neurodegenerative diseases. METHODS: To investigate the role of systemic inflammation in the progression of neuronal TDP-43 pathology, AAV9 particles driven by the UCHL1 promoter were delivered to the frontal cortex of wild-type aged mice via intracranial injections to overexpress TDP-43 or green fluorescent protein (GFP) in corticospinal motor neurons. Animals were then subjected to a low-dose (500 μg/kg) intraperitoneal E. coli lipopolysaccharide (LPS) administration challenge for 2 weeks to mimic a chronically altered low-grade systemic inflammatory state. Mice were then subjected to neurobehavioral studies, followed by biochemical and immunohistochemical analyses of the brain tissue. RESULTS: In the present study, we report that elevated neuronal TDP-43 levels induced microglial and astrocytic activation in the cortex of injected mice followed by increased RANTES signaling. Moreover, overexpression of TDP-43 exerted abundant mouse immunoglobulin G (IgG), CD3, and CD4+ T cell infiltration as well as endothelial and pericyte activation suggesting increased blood-brain barrier permeability. The BBB permeability in TDP-43 overexpressing brains yielded the frontal cortex vulnerable to the systemic inflammatory response following LPS treatment, leading to marked neutrophil infiltration, neuronal loss, reduced synaptosome-associated protein 25 (SNAP-25) levels, and behavioral impairments in the radial arm water maze (RAWM) task. CONCLUSIONS: These results reveal a novel role for TDP-43 in BBB permeability and leukocyte recruitment, indicating complex intermolecular interactions between an altered systemic inflammatory state and pathologically prone TDP-43 protein to promote disease progression

Histone deacetylase 6 inhibition improves memory and reduces total tau levels in a mouse model of tau deposition

INTRODUCTION: Tau pathology is associated with a number of age-related neurodegenerative disorders. Few treatments have been demonstrated to diminish the impact of tau pathology in mouse models and none are yet effective in humans. Histone deacetylase 6 (HDAC6) is an enzyme that removes acetyl groups from cytoplasmic proteins, rather than nuclear histones. Its substrates include tubulin, heat shock protein 90 and cortactin. Tubastatin A is a selective inhibitor of HDAC6. Modification of tau pathology by specific inhibition of HDAC6 presents a potential therapeutic approach in tauopathy. METHODS: We treated rTg4510 mouse models of tau deposition and non-transgenic mice with tubastatin (25 mg/kg) or saline (0.9%) from 5 to 7 months of age. Cognitive behavior analysis, histology and biochemical analysis were applied to access the effect of tubastatin on memory, tau pathology and neurodegeneration (hippocampal volume). RESULTS: We present data showing that tubastatin restored memory function in rTg4510 mice and reversed a hyperactivity phenotype. We further found that tubastatin reduced the levels of total tau, both histologically and by western analysis. Reduction in total tau levels was positively correlated with memory improvement in these mice. However, there was no impact on phosphorylated forms of tau, either by histology or western analysis, nor was there an impact on silver positive inclusions histologically. CONCLUSION: Potential mechanisms by which HDAC6 inhibitors might benefit the rTg4510 mouse include stabilization of microtubules secondary to increased tubulin acetylation, increased degradation of tau secondary to increased acetylation of HSP90 or both. These data support the use of HDAC6 inhibitors as potential therapeutic agents against tau pathology

CCL2 Overexpression in the Brain Promotes Glial Activation and Accelerates Tau Pathology in a Mouse Model of Tauopathy

Innate immune activation is a major contributor to Alzheimer’s Disease (AD) pathophysiology, although the mechanisms involved are poorly understood. Chemokine C-C motif ligand (CCL) 2 is produced by neurons and glial cells and is upregulated in the AD brain. Transgene expression of CCL2 in mouse models of amyloidosis produces microglia-induced amyloid β oligomerization, a strong indication of the role of these activation pathways in the amyloidogenic processes of AD. We have previously shown that CCL2 polarizes microglia in wild type mice. However, how CCL2 signaling contributes to tau pathogenesis remains unknown. To address this question, CCL2 was delivered via recombinant adeno-associated virus serotype 9 into both cortex and hippocampus of a mouse model with tau pathology (rTg4510). We report that CCL2 overexpression aggravated tau pathology in rTg4510 as shown by the increase in Gallyas stained neurofibrillary tangles as well as phosphorylated tau-positive inclusions. In addition, biochemical analysis showed a reduction in the levels of detergent-soluble tau species followed by increase in the insoluble fraction, indicating a shift toward larger tau aggregates. Indeed, increased levels of high molecular weight species of phosphorylated tau were found in the mice injected with CCL2. We also report that worsening of tau pathology following CCL2 overexpression was accompanied by a distinct inflammatory response. We report an increase in leukocyte common antigen (CD45) and Cluster of differentiation 68 (CD68) expression in the brain of rTg4510 mice without altering the expression levels of a cell-surface protein Transmembrane Protein 119 (Tmem119) and ionized calcium-binding adaptor molecule 1 (Iba-1) in resident microglia. Furthermore, the analysis of cytokines in brain extract showed a significant increase in interleukin (IL)-6 and CCL3, while CCL5 levels were decreased in CCL2 mice. No changes were observed in IL-1α, IL-1β, TNF-α. IL-4, Vascular endothelial growth factor-VEGF, IL-13 and CCL11. Taken together our data report for the first time that overexpression of CCL2 promotes the increase of pathogenic tau species and is associated with glial neuroinflammatory changes that are deleterious. We propose that these events may contribute to the pathogenesis of Alzheimer’s disease and other tauopathies

Myeloid Arginase 1 Insufficiency Exacerbates Amyloid-β Associated Neurodegenerative Pathways and Glial Signatures in a Mouse Model of Alzheimer’s Disease: A Targeted Transcriptome Analysis

Brain myeloid cells, include infiltrating macrophages and resident microglia, play an essential role in responding to and inducing neurodegenerative diseases, such as Alzheimer’s disease (AD). Genome-wide association studies (GWAS) implicate many AD casual and risk genes enriched in brain myeloid cells. Coordinated arginine metabolism through arginase 1 (Arg1) is critical for brain myeloid cells to perform biological functions, whereas dysregulated arginine metabolism disrupts them. Altered arginine metabolism is proposed as a new biomarker pathway for AD. We previously reported Arg1 deficiency in myeloid biased cells using lysozyme M (LysM) promoter-driven deletion worsened amyloidosis-related neuropathology and behavioral impairment. However, it remains unclear how Arg1 deficiency in these cells impacts the whole brain to promote amyloidosis. Herein, we aim to determine how Arg1 deficiency driven by LysM restriction during amyloidosis affects fundamental neurodegenerative pathways at the transcriptome level. By applying several bioinformatic tools and analyses, we found that amyloid-β (Aβ) stimulated transcriptomic signatures in autophagy-related pathways and myeloid cells’ inflammatory response. At the same time, myeloid Arg1 deficiency during amyloidosis promoted gene signatures of lipid metabolism, myelination, and migration of myeloid cells. Focusing on Aβ associated glial transcriptomic signatures, we found myeloid Arg1 deficiency up-regulated glial gene transcripts that positively correlated with Aβ plaque burden. We also observed that Aβ preferentially activated disease-associated microglial signatures to increase phagocytic response, whereas myeloid Arg1 deficiency selectively promoted homeostatic microglial signature that is non-phagocytic. These transcriptomic findings suggest a critical role for proper Arg1 function during normal and pathological challenges associated with amyloidosis. Furthermore, understanding pathways that govern Arg1 metabolism may provide new therapeutic opportunities to rebalance immune function and improve microglia/macrophage fitness

Arginase 1 Insufficiency Precipitates Amyloid-\u3cem\u3eβ\u3c/em\u3e Deposition and Hastens Behavioral Impairment in a Mouse Model of Amyloidosis

Alzheimer’s disease (AD) includes several hallmarks comprised of amyloid-β (Aβ) deposition, tau neuropathology, inflammation, and memory impairment. Brain metabolism becomes uncoupled due to aging and other AD risk factors, which ultimately lead to impaired protein clearance and aggregation. Increasing evidence indicates a role of arginine metabolism in AD, where arginases are key enzymes in neurons and glia capable of depleting arginine and producing ornithine and polyamines. However, currently, it remains unknown if the reduction of arginase 1 (Arg1) in myeloid cell impacts amyloidosis. Herein, we produced haploinsufficiency of Arg1 by the hemizygous deletion in myeloid cells using Arg1fl/fl and LysMcreTg/+ mice crossed with APP Tg2576 mice. Our data indicated that Arg1 haploinsufficiency promoted Aβ deposition, exacerbated some behavioral impairment, and decreased components of Ragulator-Rag complex involved in mechanistic target of rapamycin complex 1 (mTORC1) signaling and autophagy. Additionally, Arg1 repression and arginine supplementation both impaired microglial phagocytosis in vitro. These data suggest that proper function of Arg1 and arginine metabolism in myeloid cells remains essential to restrict amyloidosis

Epitope analysis following active immunization with tau proteins reveals immunogens implicated in tau pathogenesis

Abstract Background Abnormal tau hyperphosphorylation and its accumulation into intra-neuronal neurofibrillary tangles are linked to neurodegeneration in Alzheimer’s disease and similar tauopathies. One strategy to reduce accumulation is through immunization, but the most immunogenic tau epitopes have so far remained unknown. To fill this gap, we immunized mice with recombinant tau to build a map of the most immunogenic tau epitopes. Methods Non-transgenic and rTg4510 tau transgenic mice aged 5 months were immunized with either human wild-type tau (Wt, 4R0N) or P301L tau (4R0N). Each protein was formulated in Quil A adjuvant. Sera and splenocytes of vaccinated mice were collected to assess the humoral and cellular immune responses to tau. We employed a peptide array assay to identify the most effective epitopes. Brain histology was utilized to measure the effects of vaccination on tau pathology and inflammation. Results Humoral immune responses following immunization demonstrated robust antibody titers (up to 1:80,000 endpoint titers) to each tau species in both mice models. The number of IFN-γ producing T cells and their proliferation were also increased in splenocytes from immunized mice, indicating an increased cellular immune response, and tau levels and neuroinflammation were both reduced. We identified five immunogenic motifs within either the N-terminal (9-15 and 21-27 amino acids), proline rich (168-174 and 220-228 amino acids), or the C-terminal regions (427-438 amino acids) of the wild-type and P301L tau protein sequence. Conclusions Our study identifies five previously unknown immunogenic motifs of wild-type and mutated (P301L) tau protein. Immunization with both proteins resulted in reduced tau pathology and neuroinflammation in a tau transgenic model, supporting the efficacy of tau immunotherapy in tauopathy.http://deepblue.lib.umich.edu/bitstream/2027.42/109522/1/12974_2014_Article_152.pd

Aberrant \u3ci\u3eAZIN2\u3c/i\u3e and Polyamine Metabolism Precipitates Tau Neuropathology

Tauopathies display a spectrum of phenotypes from cognitive to affective behavioral impairments; however, mechanisms promoting tau pathology and how tau elicits behavioral impairment remain unclear. We report a unique interaction between polyamine metabolism, behavioral impairment, and tau fate. Polyamines are ubiquitous aliphatic molecules that support neuronal function, axonal integrity, and cognitive processing. Transient increases in polyamine metabolism hallmark the cell’s response to various insults, known as the polyamine stress response (PSR). Dysregulation of gene transcripts associated with polyamine metabolism in Alzheimer’s disease (AD) brains were observed, and we found that ornithine decarboxylase antizyme inhibitor 2 (AZIN2) increased to the greatest extent. We showed that sustained AZIN2 overexpression elicited a maladaptive PSR in mice with underlying tauopathy (MAPT P301S; PS19). AZIN2 also increased acetylpolyamines, augmented tau deposition, and promoted cognitive and affective behavioral impairments. Higher-order polyamines displaced microtubule-associated tau to facilitate polymerization but also decreased tau seeding and oligomerization. Conversely, acetylpolyamines promoted tau seeding and oligomers. These data suggest that tauopathies launch an altered enzymatic signature that endorses a feed-forward cycle of disease progression. Taken together, the tau-induced PSR affects behavior and disease continuance, but may also position the polyamine pathway as a potential entry point for plausible targets and treatments of tauopathy, including AD