Novel App knock-in mouse model shows key features of amyloid pathology and reveals profound metabolic dysregulation of microglia.

BACKGROUND: 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. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA 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 Aß 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. RESULTS: 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 Aß content. 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 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. DISCUSSION: Our findings demonstrate that fibrillar Aß 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.

Preclinical and clinical evaluation of the LRRK2 inhibitor DNL201 for Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic risk factors for Parkinson's disease (PD). Increased LRRK2 kinase activity is thought to impair lysosomal function and may contribute to the pathogenesis of PD. Thus, inhibition of LRRK2 is a potential disease-modifying therapeutic strategy for PD. DNL201 is an investigational, first-in-class, CNS-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor. In preclinical models, DNL201 inhibited LRRK2 kinase activity as evidenced by reduced phosphorylation of both LRRK2 at serine-935 (pS935) and Rab10 at threonine-73 (pT73), a direct substrate of LRRK2. Inhibition of LRRK2 by DNL201 demonstrated improved lysosomal function in cellular models of disease, including primary mouse astrocytes and fibroblasts from patients with Gaucher disease. Chronic administration of DNL201 to cynomolgus macaques at pharmacologically relevant doses was not associated with adverse findings. In phase 1 and phase 1b clinical trials in 122 healthy volunteers and in 28 patients with PD, respectively, DNL201 at single and multiple doses inhibited LRRK2 and was well tolerated at doses demonstrating LRRK2 pathway engagement and alteration of downstream lysosomal biomarkers. Robust cerebrospinal fluid penetration of DNL201 was observed in both healthy volunteers and patients with PD. These data support the hypothesis that LRRK2 inhibition has the potential to correct lysosomal dysfunction in patients with PD at doses that are generally safe and well tolerated, warranting further clinical development of LRRK2 inhibitors as a therapeutic modality for PD.

Enhanced tyrosine hydroxylase activity induces oxidative stress, causes accumulation of autotoxic catecholamine metabolites, and augments amphetamine effects in vivo.

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 H2 O2 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.

Membrane transporters as mediators of synaptic dopamine dynamics: implications for disease.

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.

D1 dopamine receptor coupling to PLCß regulates forward locomotion in mice.

Several studies have reported the coupling of dopamine signaling to phospholipase C ß (PLCß) 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 PLCß, 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 PLCß pathway in the regulation of forward locomotion via dopamine receptors.

Comparison of the cost of hospitalization for respiratory syncytial virus disease versus palivizumab prophylaxis in Canadian Inuit infants.

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.

The cost-effectiveness of palivizumab for respiratory syncytial virus prophylaxis in premature infants with a gestational age of 32-35 weeks: a Canadian-based analysis .

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.