Dopamine was first identified as a neurotransmitter localized to the midbrain over 50 years ago. The dopamine transporter (DAT; SLC6A3) and the vesicular monoamine transporter 2 (VMAT2; SLC18A2) are ...regulators of dopamine homeostasis in the presynaptic neuron. DAT transports dopamine from the extracellular space into the cytosol of the presynaptic terminal. VMAT2 then packages this cytosolic dopamine into vesicular compartments for subsequent release upon neurotransmission. Thus, DAT and VMAT2 act in concert to move the transmitter efficiently throughout the neuron. Accumulation of dopamine in the neuronal cytosol can trigger oxidative stress and neurotoxicity, suggesting that the proper compartmentalization of dopamine is critical for neuron function and risk of disease. For decades, studies have examined the effects of reduced transporter function in mice (e.g. DAT‐KO, VMAT2‐KO, VMAT2‐deficient). However, we have only recently been able to assess the effects of elevated transporter expression using BAC transgenic methods (DAT‐tg, VMAT2‐HI mice). Complemented with in vitro work and neurochemical techniques to assess dopamine compartmentalization, a new focus on the importance of transporter proteins as both models of human disease and potential drug targets has emerged. Here, we review the importance of DAT and VMAT2 function in the delicate balance of neuronal dopamine.
This review describes the importance of the dopamine transporter (DAT) and the vesicular monoamine transporter 2 (VMAT2) on dopamine compartmentalization and neuronal health (A–C). While theoretical, this schematic highlights emerging evidence from mouse models of varying transporter levels. We predict that the continuum of transporter function in these animal models will allow for new discoveries concerning endogenous dopamine handling, pharmacological manipulation of the transporters, and dopamine‐dependent behaviors (D).
In Parkinson's disease, dopamine‐containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in dopamine biosynthesis. TH increases in vitro ...formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build‐up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH‐over‐expressing mice (TH‐HI) using a BAC‐transgenic approach that results in over‐expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH‐HI mice had a 3‐fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH‐HI mice showed increased striatal production of H2O2 and reduced glutathione levels. In addition, TH‐HI mice had elevated striatal levels of the neurotoxic dopamine metabolites 3,4‐dihydroxyphenylacetaldehyde and 5‐S‐cysteinyl‐dopamine and were more susceptible than wild‐type mice to the effects of amphetamine and methamphetamine. These results demonstrate that increased TH alone is sufficient to produce oxidative stress in vivo, build up autotoxic dopamine metabolites, and augment toxicity.
This paper investigates the effect of increased activity of tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine synthesis, in a novel line of TH over‐expressing mice. Past studies suggest that in synucleinopathies, early pathological changes can result in decreased TH regulation and increased activity. Here, we show that increased TH activity is sufficient to increase H2O2 and elevate levels of cysteinylated dopamine (Cys‐DA) and 3,4‐dihydroxyphenylacetaldehyde (DOPAL)—respective autotoxic products of enzymatic and spontaneous dopamine oxidation—coincident with increased dopamine turnover. These findings suggest that TH dysregulation could present a source of dopamine‐related oxidative stress unique to cells most vulnerable in Parkinson's disease. This article is accompanied by an Editorial Highlight by Elisa Greggio (https://doi.org/10.1111/jnc.15442).
Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on ...overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain.
We engineered a novel App knock-in mouse model (App
) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-β pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous App
mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aβ content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays.
Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aβ content. The App
knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged.
Our findings demonstrate that fibrillar Aβ in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.
Several studies have reported the coupling of dopamine signaling to phospholipase C β (PLCβ) both in vitro and in vivo. However, the precise physiological relevance of this signaling pathway in ...mediating dopamine behaviors is still unclear. Here we report that stimulation of dopamine receptor signaling in vivo with systemic administration of apomorphine, amphetamine, and cocaine leads to increased production of inositol triphosphate (IP3) in the mouse striatum. Using selective antagonists and dopamine D1 and D2 receptor knock-out animals, we show that the production of IP3 is mediated by the D1 receptor, but not the D2 receptor. A selective blocker of PLCβ, U73122, was used to assess the physiological relevance of D1-mediated IP3 production. We show that U73122 inhibits the locomotor-stimulating effects of apomorphine, amphetamine, cocaine, and SKF81297. Furthermore, U73122 also suppresses the spontaneous hyperactivity exhibited by dopamine transporter knock-out mice. Importantly, the effects of U73122 are selective to dopamine-mediated hyperactivity, as this compound does not affect hyperactivity induced by the glutamate NMDA receptor antagonist MK801. Finally, we present evidence showing that an imbalance of D1- and D2-mediated signaling following U73122 treatment modifies the locomotor output of animals from horizontal locomotor activity to vertical activity, further highlighting the importance of the PLCβ pathway in the regulation of forward locomotion via dopamine receptors.
Background
Microglial dysfunction is believed to play a pathogenic role in Alzheimer’s disease (AD). Microglia respond to various pathogenic drivers of AD, including amyloid‐ß (Aß) and tau, but the ...mechanisms by which microglia may become dysfunctional and contribute to disease remain unclear. Here, we aim to characterize the amyloid‐ß related pathology and microglial responses in an engineered APP knock‐in mouse model of familial AD (AppSAA).
Method
To circumvent the numerous limitations inherent to transgenesis, we used a knock‐in strategy to humanize the Aß sequence of the murine App gene and introduced three FAD mutations ‐ Swedish (KM670/671NL), Arctic (E693G) and Austrian (T712I) ‐ using homologous recombination. We then conducted an extensive biochemical and histological characterization of various tissues from the three resulting genotypes at various ages. Finally, we conducted a deep‐phenotyping analysis of brain‐sorted microglia using multi‐omics approaches.
Result
The AppSAA knock‐in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered glial responses and increased levels of markers of neurodegeneration such as CSF Tau and neurofilament light chain. We found lipid accumulation and an exacerbated disease‐associated transcriptomic response in methoxy‐X04‐positive, phagocytic microglia.
Conclusion
Altogether, our in‐depth analysis of the AppSAA knock‐in mouse model confirms emergence of disease‐relevant biology and progressive accumulation of pathological hallmarks of AD. Our data lends further support to the notion that phagocytic microglia undergo profound cellular alterations, including lysosomal dysfunction, lipid dyshomeostasis, and other metabolic changes. Since this new mouse model can be used to investigate multiple relevant aspects of AD biology, we have made it broadly available to the scientific community.
ABSTRACT
Background: Prophylactic therapy with palivizumab, a humanized monoclonal antibody, has been shown to reduce the number of respiratory syncytial virus (RSV)-related hospitalizations in ...preterm infants, including those in the 32-35 weeks' gestational age (GA) subgroup. The cost-effectiveness of this therapy in Canada is unknown.
Objectives: To evaluate the cost-effectiveness of palivizumab as respiratory syncytial virus prophylaxis in premature infants born at 32-35 weeks' GA.
Design: A decision analytic model was designed to compare both direct and indirect medical costs and benefits of prophylaxis in this subgroup of premature infants. Sensitivity analyses were performed to ascertain the robustness of the model for five point estimates: mortality rate, discounting rates, health-utility values, degree of vial-sharing and administration costs. A probabilistic sensitivity analysis (PSA) was also conducted.
Setting: Canadian publicly funded health-care system (Ministry of Health payer perspective) for base-case analysis. Societal perspective, accounting for future lost productivity, was adopted for a secondary analysis.
Participants: Canadian infants born at 32-35 weeks' GA without chronic lung disease.
Interventions: Palivizumab prophylaxis versus no prophylaxis.
Main outcome measures: Expected costs and incremental cost-effectiveness ratio expressed as cost per life-year gained (LYG) and quality-adjusted life-year (QALY) using 2007 Canadian dollars.
Results: The expected costs were higher for palivizumab prophylaxis as compared with no prophylaxis. The incremental cost-effectiveness ratio (ICER) for the base-case scenario was $20 924 per QALY after discounting, which is considered cost-effective in Canada. When the uncertainty of the input parameter assumptions was tested through sensitivity analyses assessing several data sources for five key parameters, no substantial differences were found from the base-case results. The PSA indicated a 0.99 probability that the ICER for palivizumab was less than $50 000/QALY. Sub-analyses that varied the number of risk factors found that for infants with two or more risk factors, or at least moderate risk, palivizumab had incremental costs per QALY that indicated moderate-to-strong evidence for adoption (range: $808-81 331, per QALY).
Conclusions: Palivizumab was cost-effective and the authors' model supports prophylaxis for infants born at 32-35 weeks' GA, particularly those with more than two risk factors or at least a moderate level of risk according to a risk scoring tool.
BACKGROUND:The objectives were to compare actual respiratory syncytial virus (RSV) hospitalization rates and costs in a cohort of Inuit infants to hypothetical palivizumab prophylaxis strategies for ...infants of all gestational ages in the Eastern Canadian Arctic.
METHODS:Incidence and costs of RSV hospitalization were collected for infants admitted to the Baffin Regional Hospital in 2002, before the initiation of palivizumab. There was a comparison of the actual costs to the costs associated with 8 palivizumab strategies stratified by age (<6 months, <1 year) and location (overall, town Iqaluit, rural communities). It was assumed that each category would receive universal palivizumab prophylaxis resulting in a 78% decrease in RSV admissions. The net costs incurred, number needed to treat (NNT), and incremental costs per hospitalization avoided were calculated for each comparison.
RESULTS:There was a great variation in the rates and costs associated with RSV admissions between Iqaluit and the communities. For infants <1 year of age residing in Iqaluit, the mean admission cost was $3915, and palivizumab prophylaxis had an NNT of 20.4 and cost of $162,551 per admission avoided. For rural infants <6 months, the mean cost of admission was $23,030, and palivizumab prophylaxis resulted in an NNT of 3.9 to 2.5 and cost savings of up to $8118 per admission avoided.
CONCLUSIONS:Due to the high rates and costs associated with RSV admissions, administration of palivizumab in rural communities in the Canadian Arctic to infants less than 6 months of age could result in net cost savings.
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