The Arylamine N-Acetyltransferases as Therapeutic Targets in Metabolic Diseases Associated with Mitochondrial Dysfunction.

In humans, there are two arylamine N-acetyltransferase genes that encode functional enzymes (NAT1 and NAT2) as well as one pseudogene, all of which are located together on chromosome 8. Although they were first identified by their role in the acetylation of drugs and other xenobiotics, recent studies have shown strong associations for both enzymes in a variety of diseases, including cancer, cardiovascular disease, and diabetes. There is growing evidence that this association may be causal. Consistently, NAT1 and NAT2 are shown to be required for healthy mitochondria. This review discusses the current literature on the role of both NAT1 and NAT2 in mitochondrial bioenergetics. It will attempt to relate our understanding of the evolution of the two genes with biologic function and then present evidence that several major metabolic diseases are influenced by NAT1 and NAT2. Finally, it will discuss current and future approaches to inhibit or enhance NAT1 and NAT2 activity/expression using small-molecule drugs. SIGNIFICANCE STATEMENT: The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 share common features in their associations with mitochondrial bioenergetics. This review discusses mitochondrial function as it relates to health and disease, and the importance of NAT in mitochondrial function and dysfunction. It also compares NAT1 and NAT2 to highlight their functional similarities and differences. Both NAT1 and NAT2 are potential drug targets for diseases where mitochondrial dysfunction is a hallmark of onset and progression.

Characterization of the skeletal muscle arginine methylome in health and disease reveals remodeling in amyotrophic lateral sclerosis.

Arginine methylation is a protein posttranslational modification important for the development of skeletal muscle mass and function. Despite this, our understanding of the regulation of arginine methylation under settings of health and disease remains largely undefined. Here, we investigated the regulation of arginine methylation in skeletal muscles in response to exercise and hypertrophic growth, and in diseases involving metabolic dysfunction and atrophy. We report a limited regulation of arginine methylation under physiological settings that promote muscle health, such as during growth and acute exercise, nor in disease models of insulin resistance. In contrast, we saw a significant remodeling of asymmetric dimethylation in models of atrophy characterized by the loss of innervation, including in muscle biopsies from patients with myotrophic lateral sclerosis (ALS). Mass spectrometry-based quantification of the proteome and asymmetric arginine dimethylome of skeletal muscle from individuals with ALS revealed the largest compendium of protein changes with the identification of 793 regulated proteins, and novel site-specific changes in asymmetric dimethyl arginine (aDMA) of key sarcomeric and cytoskeletal proteins. Finally, we show that in vivo overexpression of PRMT1 and aDMA resulted in increased fatigue resistance and functional recovery in mice. Our study provides evidence for asymmetric dimethylation as a regulator of muscle pathophysiology and presents a valuable proteomics resource and rationale for numerous methylated and nonmethylated proteins, including PRMT1, to be pursued for therapeutic development in ALS.

Associations of postprandial ghrelin, liver-expressed antimicrobial peptide 2 and leptin levels with body composition, disease progression and survival in patients with amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Loss of appetite contributes to weight loss and faster disease progression in amyotrophic lateral sclerosis (ALS). Impairment of appetite control in ALS may include altered production or action of orexigenic (i.e., ghrelin) and anorexigenic (i.e., liver-expressed antimicrobial peptide 2 [LEAP2] and leptin) hormones. We aimed to determine if postprandial circulating ghrelin levels, LEAP2 levels, LEAP2:ghrelin molar ratio and leptin levels differ in ALS patients compared to non-neurodegenerative disease controls, and whether they are associated with disease progression and body composition. METHODS: In this prospective natural history study, we assessed postprandial plasma levels of ghrelin, LEAP2 and leptin in patients with ALS (cases; n = 46) and controls (controls; n = 43). For cases, measures were compared to changes in body weight, body composition and clinical outcomes. RESULTS: Postprandial ghrelin level was decreased by 52% in cases compared to controls (p = 0.013). LEAP2:ghrelin molar ratio was increased by 249% (p = 0.009), suggesting greater ghrelin resistance. Patients with lower LEAP2:ghrelin tended to have better functional capacity at assessment, as inferred by the ALS Functional Rating Scale-Revised (τ = -0.179, p = 0.086). Furthermore, ghrelin and LEAP2:ghrelin molar ratio correlated with diagnostic delay (ghrelin, τ = 0.223, p = 0.029; LEAP2:ghrelin, τ = -0.213, p = 0.037). Baseline ghrelin level, LEAP2 level, LEAP2:ghrelin ratio and leptin level were, however, not predictive of change in functional capacity during follow-up. Also, patients with higher postprandial ghrelin levels (hazard ratio [HR] 1.375, p = 0.048), and lower LEAP2:ghelin ratios (HR 0.828, p = 0.051) had an increased risk of earlier death. CONCLUSIONS: Reduced postprandial ghrelin levels, coupled with increased LEAP2:ghrelin molar ratios, suggests a loss of ghrelin action in patients with ALS. Given ghrelin's actions on appetite, metabolism and neuroprotection, reduced ghrelin and greater ghrelin resistance could contribute to impaired capacity to tolerate the physiological impact of disease. Comprehensive studies are needed to explain how ghrelin and LEAP2 contribute to body weight regulation and disease progression in ALS.

Skeletal muscle in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS), the major adult-onset motor neuron disease, has been viewed almost exclusively as a disease of upper and lower motor neurons, with muscle changes interpreted as a consequence of the progressive loss of motor neurons and neuromuscular junctions. This has led to the prevailing view that the involvement of muscle in ALS is only secondary to motor neuron loss. Skeletal muscle and motor neurons reciprocally influence their respective development and constitute a single functional unit. In ALS, multiple studies indicate that skeletal muscle dysfunction might contribute to progressive muscle weakness, as well as to the final demise of neuromuscular junctions and motor neurons. Furthermore, skeletal muscle has been shown to participate in disease pathogenesis of several monogenic diseases closely related to ALS. Here, we move the narrative towards a better appreciation of muscle as a contributor of disease in ALS. We review the various potential roles of skeletal muscle cells in ALS, from passive bystanders to active players in ALS pathophysiology. We also compare ALS to other motor neuron diseases and draw perspectives for future research and treatment.

Lower hypothalamic volume with lower body mass index is associated with shorter survival in patients with amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Weight loss in patients with amyotrophic lateral sclerosis (ALS) is associated with faster disease progression and shorter survival. Decreased hypothalamic volume is proposed to contribute to weight loss due to loss of appetite and/or hypermetabolism. We aimed to investigate the relationship between hypothalamic volume and body mass index (BMI) in ALS and Alzheimer's disease (AD), and the associations of hypothalamic volume with weight loss, appetite, metabolism and survival in patients with ALS. METHODS: We compared hypothalamic volumes from magnetic resonance imaging scans with BMI for patients with ALS (n = 42), patients with AD (n = 167) and non-neurodegenerative disease controls (n = 527). Hypothalamic volumes from patients with ALS were correlated with measures of appetite and metabolism, and change in anthropomorphic measures and disease outcomes. RESULTS: Lower hypothalamic volume was associated with lower and higher BMI in ALS (quadratic association; probability of direction = 0.96). This was not observed in AD patients or controls. Hypothalamic volume was not associated with loss of appetite (p = 0.58) or hypermetabolism (p = 0.49). Patients with lower BMI and lower hypothalamic volume tended to lose weight (p = 0.08) and fat mass (p = 0.06) over the course of their disease, and presented with an increased risk of earlier death (hazard ratio [HR] 3.16, p = 0.03). Lower hypothalamic volume alone trended for greater risk of earlier death (HR 2.61, p = 0.07). CONCLUSION: These observations suggest that lower hypothalamic volume in ALS contributes to positive and negative energy balance, and  is not universally associated with loss of appetite or hypermetabolism. Critically, lower hypothalamic volume with lower BMI was associated with weight loss and earlier death.

Venous creatinine as a biomarker for loss of fat-free mass and disease progression in patients with amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: To establish the utility of venous creatinine as a biomarker to monitor loss of fat-free mass in patients with amyotrophic lateral sclerosis (ALS). METHODS: In this multicenter natural history study, body composition and venous creatinine were assessed in 107 patients with ALS and 52 healthy controls. Longitudinal patterns of venous creatinine and its association with the risk of death during follow-up were determined in a cohort of patients with ALS from Australia (n = 69) and the Netherlands (n = 38). RESULTS: The mean levels of venous creatinine were 75.78 ± 11.15 µmol/L for controls, 70.25 ± 12.81 µmol/L for Australian patients, and 59.95 ± 14.62 µmol/L for Dutch patients with ALS. The relationship between measures of venous creatinine and fat-free mass was similar between all groups (r = 0.36, p < 0.001). Within patients, fat-free mass declined by 0.31 (95% confidence interval [CI]: 0.22-0.40) kg/month, and venous creatinine declined by 0.52 (95% CI: 0.38-0.66) µmol/L/month, with a longitudinal correlation of 0.57 (95% CI: 0.35-0.76, p < 0.001). Lower levels of venous creatinine were associated with increased risk for earlier death in patients with ALS (hazard ratio = 0.94, 95% CI: 0.90-0.98, p = 0.007). CONCLUSIONS: Venous creatinine is decreased in ALS and declines alongside a decline in fat-free mass over the course of the disease, and may serve as a practical marker to monitor the change of fat-free mass in patients with ALS. This could inform clinical care and provide an alternative endpoint for the evaluation of therapeutic interventions that focus on slowing the loss of fat-free mass and disease progression in ALS.

A Road Map for Remote Digital Health Technology for Motor Neuron Disease.

Despite recent and potent technological advances, the real-world implementation of remote digital health technology in the care and monitoring of patients with motor neuron disease has not yet been realized. Digital health technology may increase the accessibility to and personalization of care, whereas remote biosensors could optimize the collection of vital clinical parameters, irrespective of patients' ability to visit the clinic. To facilitate the wide-scale adoption of digital health care technology and to align current initiatives, we outline a road map that will identify clinically relevant digital parameters; mediate the development of benefit-to-burden criteria for innovative technology; and direct the validation, harmonization, and adoption of digital health care technology in real-world settings. We define two key end products of the road map: (1) a set of reliable digital parameters to capture data collected under free-living conditions that reflect patient-centric measures and facilitate clinical decision making and (2) an integrated, open-source system that provides personalized feedback to patients, health care providers, clinical researchers, and caregivers and is linked to a flexible and adaptable platform that integrates patient data in real time. Given the ever-changing care needs of patients and the relentless progression rate of motor neuron disease, the adoption of digital health care technology will significantly benefit the delivery of care and accelerate the development of effective treatments.

C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

The canonical complement component 5a (C5a) receptor (C5aR) 1 has well-described roles in tumorigenesis but the contribution of the second receptor, C5aR2, is unclear. The present study demonstrates that B16.F0 melanoma cells express mRNA for both C5aR1 and C5aR2 and signal through ERK and p38 MAPKs in response to C5a. Despite this, C5a had no impact on melanoma cell proliferation or migration in vitro. In vivo studies demonstrated that the growth of B16.F0 melanoma tumors was increased in C5aR2-/- mice but reduced in C5aR1-/- mice and wild-type mice treated with a C5aR1 antagonist. Analysis of tumor-infiltrating leukocyte populations showed no significant differences between wild-type and C5aR2-/- mice. Conversely, percentages of myeloid-derived suppressor cells, macrophages, and regulatory T lymphocytes were lower in tumors from C5aR1-/- mice, whereas total (CD3+) T lymphocytes and CD4+ subsets were higher. Analysis of cytokine and chemokine levels also showed plasma IFN-γ was higher and tumor C-C motif chemokine ligand 2 was lower in the absence of C5aR1. The results suggest that C5aR1 signaling supports melanoma growth by promoting infiltration of immunosuppressive leukocyte populations into the tumor microenvironment, whereas C5aR2 has a more restricted but beneficial role in limiting tumor growth. Overall, these data support the potential of C5aR1-inhibitory therapies for melanoma.-Nabizadeh, J. A., Manthey, H. D., Panagides, N., Steyn, F. J., Lee, J. D., Li, X. X., Akhir, F. N. M., Chen, W., Boyle, G. M., Taylor, S. M., Woodruff, T. M., Rolfe, B. E. C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

Hypermetabolism in ALS is associated with greater functional decline and shorter survival.

OBJECTIVE: To determine the prevalence of hypermetabolism, relative to body composition, in amyotrophic lateral sclerosis (ALS) and its relationship with clinical features of disease and survival. METHODS: Fifty-eight patients with clinically definite or probable ALS as defined by El Escorial criteria, and 58 age and sex-matched control participants underwent assessment of energy expenditure. Our primary outcome was the prevalence of hypermetabolism in cases and controls. Longitudinal changes in clinical parameters between hypermetabolic and normometabolic patients with ALS were determined for up to 12 months following metabolic assessment. Survival was monitored over a 30-month period following metabolic assessment. RESULTS: Hypermetabolism was more prevalent in patients with ALS than controls (41% vs 12%, adjusted OR=5.4; p<0.01). Change in body weight, body mass index and fat mass (%) was similar between normometabolic and hypermetabolic patients with ALS. Mean lower motor neuron score (SD) was greater in hypermetabolic patients when compared with normometabolic patients (4 (0.3) vs 3 (0.7); p=0.04). In the 12 months following metabolic assessment, there was a greater change in Revised ALS Functional Rating Scale score in hypermetabolic patients when compared with normometabolic patients (-0.68 points/month vs -0.39 points/month; p=0.01). Hypermetabolism was inversely associated with survival. Overall, hypermetabolism increased the risk of death during follow-up to 220% (HR 3.2, 95% CI 1.1 to 9.4, p=0.03). CONCLUSIONS AND RELEVANCE: Hypermetabolic patients with ALS have a greater level of lower motor neuron involvement, faster rate of functional decline and shorter survival. The metabolic index could be important for informing prognosis in ALS.

The Complement C3a Receptor Contributes to Melanoma Tumorigenesis by Inhibiting Neutrophil and CD4+ T Cell Responses.

The complement peptide C3a is a key component of the innate immune system and a major fragment produced following complement activation. We used a murine model of melanoma (B16-F0) to identify a hitherto unknown role for C3a-C3aR signaling in promoting tumor growth. The results show that the development and growth of B16-F0 melanomas is retarded in mice lacking C3aR, whereas growth of established melanomas can be arrested by C3aR antagonism. Flow cytometric analysis showed alterations in tumor-infiltrating leukocytes in the absence of C3aR. Specifically, neutrophils and CD4(+) T lymphocyte subpopulations were increased, whereas macrophages were reduced. The central role of neutrophils was confirmed by depletion experiments that reversed the tumor inhibitory effects observed in C3aR-deficient mice and returned tumor-infiltrating CD4(+) T cells to control levels. Analysis of the tumor microenvironment showed upregulation of inflammatory genes that may contribute to the enhanced antitumor response observed in C3aR-deficient mice. C3aR deficiency/inhibition was also protective in murine models of BRAF(V600E) mutant melanoma and colon and breast cancer, suggesting a tumor-promoting role for C3aR signaling in a range of tumor types. We propose that C3aR activation alters the tumor inflammatory milieu, thereby promoting tumor growth. Therapeutic inhibition of C3aR may therefore be an effective means to trigger an antitumor response in melanoma and other cancers.