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 Arg1 fl/fl and LysMcre Tg/+ 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.

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.

Tau stabilizes microtubules; however, in Alzheimer’s disease (AD) and tauopathies, tau becomes hyperphosphorylated, aggregates, and results in neuronal death. Our group recently uncovered a unique interaction between polyamine metabolism and tau fate. Polyamines exert an array of physiological effects that support neuronal function and cognitive processing. Specific stimuli can elicit a polyamine stress response (PSR), resulting in altered central polyamine homeostasis. Evidence suggests that elevations in polyamines following a short-term stressor are beneficial; however, persistent stress and subsequent PSR activation may lead to maladaptive polyamine dysregulation, which is observed in AD, and may contribute to neuropathology and disease progression. Male and female mice harboring tau P301L mutation (rTg4510) were examined for a tau-induced central polyamine stress response (tau-PSR). The direct effect of tau-PSR byproducts on tau fibrillization and oligomerization were measured using a thioflavin T assay and a N2a split superfolder GFP-Tau (N2a-ssGT) cell line, respectively. To therapeutically target the tau-PSR, we bilaterally injected caspase 3-cleaved tau truncated at aspartate 421 (AAV9 Tau ΔD421) into the hippocampus and cortex of spermidine/spermine-N1-acetyltransferase (SSAT), a key regulator of the tau-PSR, knock out (SSAT-/-), and wild type littermates, and the effects on tau neuropathology, polyamine dysregulation, and behavior were measured. Lastly, cellular models were employed to further examine how SSAT repression impacted tau biology. Tau induced a unique tau-PSR signature in rTg4510 mice, notably in the accumulation of acetylated spermidine. In vitro, higher-order polyamines prevented tau fibrillization but acetylated spermidine failed to mimic this effect and even promoted fibrillization and oligomerization. AAV9 Tau ΔD421 also elicited a unique tau-PSR in vivo, and targeted disruption of SSAT prevented the accumulation of acetylated polyamines and impacted several tau phospho-epitopes. Interestingly, SSAT knockout mice presented with altered behavior in the rotarod task, the elevated plus maze, and marble burying task, thus highlighting the impact of polyamine homeostasis within the brain. These data represent a novel paradigm linking tau pathology and polyamine dysfunction and that targeting specific arms within the polyamine pathway may serve as new targets to mitigate certain components of the tau phenotype.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by alpha-synuclein accumulation and loss of dopaminergic neurons in the substantia nigra (SN) region of the brain. Increased levels of alpha-synuclein have been shown to result in loss of mitochondrial electron transport chain complex I activity leading to increased reactive oxygen species (ROS) production. WT alpha-synuclein was stably overexpressed in human BE(2)-M17 neuroblastoma cells resulting in increased levels of an alpha-synuclein multimer, but no increase in alpha-synuclein monomer levels. Oxygen consumption was decreased by alpha-synuclein overexpression, but ATP levels did not decrease and ROS levels did not increase. Treatment with ferrous sulfate, a ROS generator, resulted in decreased oxygen consumption in both control and alpha-synuclein overexpressing cells. However, this treatment only decreased ATP levels and increased ROS production in the cells overexpressing alpha-synuclein. Similarly, paraquat, another ROS generator, decreased ATP levels in the alpha-synuclein overexpressing cells, but not in the control cells, further demonstrating how alpha-synuclein sensitized the cells to oxidative insult. Proteomic analysis yielded molecular insights into the cellular adaptations to alpha-synuclein overexpression, such as the increased abundance of many mitochondrial proteins. Many amino acids and citric acid cycle intermediates and their ester forms were individually supplemented to the cells with L-serine, L-proline, L-aspartate, or L-glutamine decreasing ROS production in oxidatively stressed alpha-synuclein overexpressing cells, while diethyl oxaloacetate or L-valine supplementation increased ATP levels. These results suggest that dietary supplementation with individual metabolites could yield bioenergetic improvements in PD patients to delay loss of dopaminergic neurons.

Currently no disease modifying agents exist on the market for Alzheimer's disease (AD). One major hallmark includes tau neuropathology and associates with several neurodegenerative diseases collectively known as tauopathies. Strategies aimed at reducing tau burden include increasing clearance, targeting kinases, posttranslational modifications, reducing aggregation, and modifying inflammation. Arginine metabolism acts as a substrate and critical branch-point, which dictates several outcomes: nitric oxide generation from nitric oxide synthases (NOS) or polyamine production from arginase (Arg1). Several reports show that nitration of tau impacts tau metabolism and increases aggregation, thus biasing one or more pathways may dictate tau fate. Depletion of arginine may also lead to increased autophagy through amino acid sensing. Utilizing gene therapy (adeno-associated virus), cellular, animal models of tauopathy (rTg4510 and PS19 mice), and recombinant protein experiments we tested each of these potential mechanisms: 1) decrease tau nitration (shifting arginine metabolism away from the NOS pathway); 2) arginine depletion (and activation of autophagy); and 3) polyamine production. We find dysregulation of the polyamine pathway in tau transgenic mice. Our data indicates that Arg1 overexpression in mouse models of tauopathy reduces the many aspects of the tau phenotype including reduced phospho-tau and nitrated tau, reduced tangle pathology, reduced atrophy, reduced high molecular weight tau, reduced markers of inflammation, reduced inhibitors of autophagy, and reduced protein kinase activation. We find similar outcomes in cell lines overexpressing tau with parallel Arg1 manipulations. Importantly, we identified that higher-order polyamines at physiological concentrations directly block tau aggregation and facilitate tubulin polymerization but acetylated forms fail to mimic this affect. These data suggest arginine metabolism and the polyamine pathway as therapeutic targets that may arrest or slow the progression of the tau pathology in models of tauopathies.

Tau accumulation remains one of the closest correlates of neuronal loss in Alzheimer's disease. In addition, tau associates with several other neurodegenerative diseases, collectively known as tauopathies, in which clinical phenotypes manifest as cognitive impairment, behavioral disturbances, and motor impairment. Polyamines act as bivalent regulators of cellular function and are involved in numerous biological processes. The regulation of the polyamines system can become dysfunctional during disease states. Arginase 1 (Arg1) and nitric oxide synthases compete forl-arginine to produce either polyamines or nitric oxide, respectively. Herein, we show that overexpression of Arg1 using adeno-associated virus (AAV) in the CNS of rTg4510 tau transgenic mice significantly reduced phospho-tau species and tangle pathology. Sustained Arg1 overexpression decreased several kinases capable of phosphorylating tau, decreased inflammation, and modulated changes in the mammalian target of rapamycin and related proteins, suggesting activation of autophagy. Arg1 overexpression also mitigated hippocampal atrophy in tau transgenic mice. Conversely, conditional deletion of Arg1 in myeloid cells resulted in increased tau accumulation relative to Arg1-sufficient mice after transduction with a recombinant AAV–tau construct. These data suggest that Arg1 and the polyamine pathway may offer novel therapeutic targets for tauopathies.

Tau pathology associates with several neurodegenerative diseases including Alzheimer's disease (AD) collectively known as tauopathies. The role of inflammation in neurodegenerative diseases remains controversial yet provides opportunity for therapeutic intervention. By-products of certain activation profiles reduce disease pathology whereas others promote it. Inhibiting one pathway along the inflammatory milieu may promote an alternative cascade. Arginase 1 (Arg1) and nitric oxide synthases (NOS) increase during certain disease states and have been implicated in AD pathogenesis. Arg1 and NOS compete for L-arginine to produce either ornithine and polyamines or nitric oxide, respectively. Polyamines act as bivalent regulators of cellular function, promoting cell growth or cell death, depending on cell type and the microenvironment. We identified dysregulation of proteins associated with polyamine synthesis and metabolism in rTg4510 tau transgenic mice compared to non-transgenic littermates. We postulate that increased Arg1 expression in the CNS impacts tau pathology. Four month-old rTg4510 tau transgenic mice receive an intracranial injection of recombinant adeno associated virus (rAAV) into the hippocampus of either rAAV-Arg1 or a control vector rAAV-GFP. Four months post injection brains were harvested for histology, immunohistochemistry, western blotting and ELISA. We show that overexpression Arg1 for a duration of four months in the hippocampus of rTg4510 tau transgenic mice using rAAV-Arg1 reduced several phospho-tau epitopes (i.e. AT8, AT180, AT270, Ser262, Ser396) and tangle pathology indicated by Gallyas silver positive staining compared the control vector rAAV-GFP (green fluorescent protein). Furthermore we found that Arg1 decreased several kinases associated with phospho-tau including phospho-GSK3 alpha 216/ beta 279 and CDK5 levels. Additionally, Arg1 overexpression decreased several cytokines and inflammatory markers including IL-1beta, TNF-alpha, IL-12, along with microglial activation measured by CD45 immunohistochemistry. Unbiased stereology showed no change in the number of neurons compared to the control vector however, Arg1 overexpression slightly but significantly mitigated hippocampal atrophy. These data suggests that Arg1 and the polyamine pathway may provide new and potential therapeutic targets for AD and tauopathies.