Mutations in FUS and TARDBP cause amyotrophic lateral sclerosis (ALS), but the precise mechanisms of selective motor neuron degeneration remain unresolved. To address if pathomechanisms are shared across mutations and related to either gain- or loss-of-function, we performed single-cell RNA sequencing across isogenic induced pluripotent stem cell-derived neuron types, harbouring FUS P525L, FUS R495X, TARDBP M337V mutations or FUS knockout. Transcriptional changes were far more pronounced in motor neurons than interneurons. About 20% of uniquely dysregulated motor neuron transcripts were shared across FUS mutations, half from gain-of-function. Most indicated mitochondrial impairments, with attenuated pathways shared with mutant TARDBP M337V as well as C9orf72-ALS patient motor neurons. Mitochondrial motility was impaired in ALS motor axons, even with nuclear localized FUS mutants, demonstrating shared toxic gain-of-function mechanisms across FUS- and TARDBP-ALS, uncoupled from protein mislocalization. These early mitochondrial dysfunctions unique to motor neurons may affect survival and represent therapeutic targets in ALS.

Brown adipose tissue (BAT) is a heater organ that expresses thermogenic uncoupling protein 1 (UCP1) to maintain high body temperatures during cold stress. BAT thermogenesis is considered an overarching mammalian trait, but its evolutionary origin is unknown. We show that adipose tissue of marsupials, which diverged from eutherian mammals ~150 million years ago, expresses a nonthermogenic UCP1 variant governed by a partial transcriptomic BAT signature similar to that found in eutherian beige adipose tissue. We found that the reconstructed UCP1 sequence of the common eutherian ancestor displayed typical thermogenic activity, whereas therian ancestor UCP1 is nonthermogenic. Thus, mammalian adipose tissue thermogenesis may have evolved in two distinct stages, with a prethermogenic stage in the common therian ancestor linking UCP1 expression to adipose tissue and thermal stress. We propose that in a second stage, UCP1 acquired its thermogenic function specifically in eutherians, such that the onset of mammalian BAT thermogenesis occurred only after the divergence from marsupials. 

Objective: Enhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms.

Method: NAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP).

Result: NAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP.

Conclusion: NAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications.

The neurotoxin MPP⁺ triggers cell death of dopamine neurons and induces Parkinson's disease symptoms in mice and men, but the immediate transcriptional response to this neurotoxin has not been studied. We therefore treated human SH-SY5Y cells with a low dose (0.1 mM) of MPP⁺ and measured the effect on nascent transcription by precision run-on sequencing (PRO-seq). We found that transcription of the mitochondrial genome was significantly reduced already after 30 min, whereas nuclear gene transcription was unaffected. Inhibition of respiratory complex I by MPP⁺ led to reduced ATP production, that may explain the diminished activity of mitochondrial RNA polymerase. Our results show that MPP⁺ has a direct effect on mitochondrial function and transcription, and that other gene expression or epigenetic changes induced by this neurotoxin are secondary effects that reflect a cellular adaptation program.

Background: Tissue-resident macrophages have mixed developmental origins. They derive in variable extent from yolk sac (YS) hematopoiesis during embryonic development. Bone marrow (BM) hematopoietic progenitors give rise to tissue macrophages in postnatal life, and their contribution increases upon organ injury. Since the phenotype and functions of macrophages are modulated by the tissue of residence, the impact of their origin and developmental paths has remained incompletely understood. Methods: In order to decipher cell-intrinsic macrophage programs, we immortalized hematopoietic progenitors from YS and BM using conditional HoxB8, and carried out an in-depth functional and molecular analysis of differentiated macrophages. Results: While YS and BM macrophages demonstrate close similarities in terms of cellular growth, differentiation, cell death susceptibility and phagocytic properties, they display differences in cell metabolism, expression of inflammatory markers and inflammasome activation. Reduced abundance of PYCARD (ASC) and CASPASE-1 proteins in YS macrophages abrogated interleukin-1 beta production in response to canonical and non-canonical inflammasome activation. Conclusions: Macrophage ontogeny is associated with distinct cellular programs and immune response. Our findings contribute to the understanding of the regulation and programming of macrophage functions.

Thermogenic adipose tissue, which comprises classical brown and beige adipose tissue, has the ability to improve systemic metabolism. Its identification in adult humans has fostered extensive investigations on the therapeutic value to counteract obesity and metabolic disorders. Sex and gender differences of human thermogenic adipose tissue, however, are still understudied despite their importance for personalized treatment options. Here, we review studies reporting human sex differences of thermogenic adipose tissue and related potential improvements of systemic energy metabolism. An increasing body of evidence suggests higher prevalence, mass and activity of thermogenic adipose tissue in women, but the consequences for metabolic disease progression and mechanisms are largely unknown. Therefore, we also discuss observations on sex-specific adipose metabolism in experimental mouse and rat studies that may assist to establish molecular mechanisms and instruct future investigations in humans.

Human fibroblast growth factor 21 (FGF21) is primarily produced and secreted by the liver as a hepatokine. This hormone circulates to its target tissues (e. g., brain, adipose tissue), which requires two components, one of the preferred FGF receptor isoforms (FGFR1c and FGFR3c) and the co-factor betaKlotho (KLB) to trigger downstream signaling pathways. Although targeting FGF21 signaling in humans by analogues and receptor agonists results in beneficial effects, e. g., improvements in plasma lipids and decreased body weight, it failed to recapitulate the improvements in glucose handling shown for many mouse models. FGF21's role and metabolic effects in mice and its therapeutic potential have extensively been reviewed elsewhere. In this review we focus on circulating FGF21 levels in humans and their associations with disease and clinical parameters, focusing primarily on obesity and obesity-associated diseases such as type-2 diabetes. We provide a comprehensive overview on human circulating FGF21 levels under normal physiology and metabolic disease. We discuss the emerging field of inactivating FGF21 in human blood by fibroblast activation protein (FAP) and its potential clinical implications.

Among obese subjects, metabolically healthy (MHO) and unhealthy obese (MUHO) subjects exist, the latter being characterized by whole-body insulin resistance, hepatic steatosis, and subclinical inflammation. Insulin resistance and obesity are known to associate with alterations in mitochondrial density, morphology, and function. Therefore, we assessed mitochondrial function in human subcutaneous preadipocytes as well as in differentiated adipocytes derived from well-matched donors. Primary subcutaneous preadipocytes from 4 insulin-resistant (MUHO) versus 4 insulin-sensitive (MHO), non-diabetic, morbidly obese Caucasians (BMI > 40 kg/m(2)), matched for sex, age, BMI, and percentage of body fat, were differentiated in vitro to adipocytes. Real-time cellular respiration was measured using an XF24 Extracellular Flux Analyzer (Seahorse). Lipolysis was stimulated by forskolin (FSK) treatment. Mitochondrial respiration was fourfold higher in adipocytes versus preadipocytes (p = 1.6*10(-9)). In adipocytes, a negative correlation of mitochondrial respiration with donors' insulin sensitivity was shown (p = 0.0008). Correspondingly, in adipocytes of MUHO subjects, an increased basal respiration (p = 0.002), higher proton leak (p = 0.04), elevated ATP production (p = 0.01), increased maximal respiration (p = 0.02), and higher spare respiratory capacity (p = 0.03) were found, compared to MHO. After stimulation with FSK, the differences in ATP production, maximal respiration and spare respiratory capacity were blunted. The differences in mitochondrial respiration between MUHO/MHO were not due to altered mitochondrial content, fuel switch, or lipid metabolism. Thus, despite the insulin resistance of MUHO, we could clearly show an elevated mitochondrial respiration of MUHO adipocytes. We suggest that the higher mitochondrial respiration reflects a compensatory mechanism to cope with insulin resistance and its consequences. Preserving this state of compensation might be an attractive goal for preventing or delaying the transition from insulin resistance to overt diabetes.

Brown adipose tissue (BAT) has been considered beneficial for metabolic health by participating in the regulation of glucose homoeostasis. The browning factors that improve glucose uptake beyond normal levels are still unknown but glucose uptake is not affected in UCP1 knockout mice. Here, we demonstrate in human white adipocytes that basal/resting glucose uptake is improved by solely elevating UCP1 protein levels. Generating human white Simpson-Golabi-Behmel syndrome (SGBS) adipocytes with a stable knockout and overexpression of UCP1, we discovered that UCP1 overexpressing adipocytes significantly improve glucose uptake by 40%. Mechanistically, this is caused by higher glycolytic flux, seen as increased oxygen consumption, extracellular acidification and lactate secretion rates. The improvements in glucose handling are comparable to white-to-brown transitions, as judged by, for the first time, directly comparing in vitro differentiated mouse brown vs white adipocytes. Although no adipogenic, metabolic and mitochondrial gene expressions were significantly altered in SGBS cells, pharmacological inhibition of GLUT1 completely abrogated differences between UCP1 + and control cells, thereby uncovering GLUT1-mediated uptake as permissive gatekeeper. Collectively, our data demonstrate that elevating UCP1 levels is sufficient to improve human white adipocytes as a glucose sink without adverse cellular effects, thus not requiring the adrenergic controlled, complex network of browning which usually hampers translational efforts.