Faculty Bibliography

Cardiolipin (CL), the signature phospholipid of mitochondria, carries four fatty acids that are remodeled after de novo synthesis. In yeast, remodeling is accomplished by the joint action of Cld1, a lipase that removes a fatty acid from CL, and Taz1, a transacylase that transfers a fatty acid from another phospholipid to monolyso-CL. While taz1 homologues have been identified in all eukaryotes, cld1 homologues have remained obscure. Here we demonstrate that ABHD18, a highly conserved protein of plants, animals, and humans, is functionally homologous to Cld1. Knockdown of Abhd18 decreased the concentration of monolyso-CL in murine, Taz-knockout myoblasts. Inactivation of Abhd18 in Drosophila substantially increased the abundance of CL. Abhd18 inactivation also reversed the increase in the rate of CL degradation, as measured with ¹³C isotopes, and the accumulation of deacylated CLs, such as monolyso-CL and dilyso-CL, in TAZ-deficient flies. CL species with more than 5 double bonds were resistant to ABHD18. Our data demonstrate that ABHD18 is the elusive lipase that hydrolyzes CL in mice and flies and presumably in other organisms. Rather than removing just one fatty acid, we show that ABHD18 deacylates CL further. Thus, ABHD18 catalyzes the breakdown of CL whereas TAZ protects CL from degradation.

UNLABELLED:Dysfunction of the endolysosomal system within neurons is a prominent feature of Alzheimer's disease (AD) pathology. Multiple AD-risk factors are known to cause hyper-activity of the early-endosome small GTPase rab5, resulting in neuronal endosomal pathway disruption. APPL1, an important rab5 effector protein, is an interface between endosomal and neuronal function through a rab5-activating interaction with the BACE1-generated C-terminal fragment (βCTF or C99) of the amyloid precursor protein (APP), a pathogenic APP fragment generated within endolysosomal compartments. To better understand the role of APPL1 in the AD endosomal phenotype, we generated a transgenic mouse model over-expressing human APPL1 within neurons (Thy1-APPL1 mice). Consistent with the important endosomal regulatory role of APPL1, Thy1-APPL1 mice have enlarged neuronal early endosomes and increased synaptic endocytosis due to increased rab5 activation. We additionally demonstrate pathological consequences of APPL1 overexpression, including functional changes in hippocampal long-term potentiation (LTP) and long-term depression (LTD), as well as degeneration of the large projection cholinergic neurons of the basal forebrain and impairment of hippocampal-dependent memory. Our findings show that increased neuronal APPL1 levels lead to a cascade of pathological effects within neurons, including early endosomal alterations, synaptic dysfunction, and neurodegeneration. Multiple risk factors and molecular regulators, including APPL1 activity, are known to contribute to the endosomal dysregulation seen in the early stages of AD, and these findings further highlight the shared pathobiology and consequences to a neuron of early endosomal pathway disruption. SIGNIFICANCE STATEMENT/UNASSIGNED:Dysfunction in the endolysosomal system within neurons is a key feature of Alzheimer's disease (AD). Multiple AD risk factors lead to hyperactivity of the early-endosome GTPase rab5, disrupting neuronal pathways including the cholinergic circuits involved early in memory decline. APPL1, a crucial rab5 effector, connects endosomal and neuronal functions through its interaction with a specific amyloid precursor protein (APP) fragment generated within endosomes. To understand APPL1's role, a transgenic mouse model over-expressing human APPL1 in neurons (Thy1-APPL1 mice) was developed. These mice show enlarged early endosomes and increased synaptic endocytosis due to rab5 activation, resulting in impaired hippocampal long-term potentiation and depression, the degeneration of basal forebrain cholinergic neurons, and memory deficits, highlighting a pathological cascade mediated through APPL1 at the early endosome.

The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. Validating the function of these candidates in vivo is challenging, due to low efficiency and low throughput of most model systems. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked-in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. We used this system to identify both cell-autonomous and non-cell autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our high-efficiency genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.

Collagen VI-related disorders (COL6-RDs) are a group of rare muscular dystrophies caused by pathogenic variants in collagen VI genes (COL6A1, COL6A2, and COL6A3). Collagen type VI is a heterotrimeric, microfibrillar component of the muscle extracellular matrix (ECM), predominantly secreted by resident fibroadipogenic precursor cells in skeletal muscle. The absence or mislocalization of collagen VI in the ECM underlies the noncell-autonomous dysfunction and dystrophic changes in skeletal muscle with a yet elusive direct mechanistic link between the ECM and myofiber dysfunction. Here, we conducted a comprehensive natural history and outcome study in a mouse model of COL6-RDs (Col6a2-/- mice) using standardized (TREAT-NMD) functional, histological, and physiological parameters. Notably, we identify a conspicuous dysregulation of the TGF-β pathway early in the disease process and propose that the collagen VI-deficient matrix is not capable of regulating the dynamic TGF-β bioavailability both at baseline and in response to muscle injury. Thus, we propose a new mechanism for pathogenesis of the disease that links the ECM regulation of TGF-β with downstream skeletal muscle abnormalities, paving the way for the development and validation of therapeutics that target this pathway.

Sodium-glucose transporter-2 inhibitor (SGLT2i) drugs are widely used for lowering blood glucose levels independent of insulin. Beyond this, these drugs induce various metabolic changes, including weight loss and impaired bone integrity. There is a significant gap in understanding SGLT2i-induced skeletal changes, as SGLT2 is not expressed in osteoblasts or osteocytes, which use glucose to remodel the bone matrix. We studied the impact of 1, 3, or 6 months of canagliflozin (CANA), an SGLT2i treatment, on the skeleton of 6-month-old genetically heterogeneous UM-HET3 mice. Significant metabolic adaptations to CANA were evident as early as 1.5 months post-treatment, specifically in male mice. CANA-treated male mice exhibited notable reductions in body weight and decreased proinflammatory and bone remodeling markers associated with reduced cortical bone remodeling indices. Bone tissue metabolome indicated enrichment in metabolites related to amino acid transport and tryptophan catabolism in CANA-treated male mice. In contrast, CANA-treated female mice showed increases in nucleic acid metabolism. An integrOmics approach of source-matched bone tissue metabolome and bone marrow RNAseq indicated a positive correlation between the two omics data sets in male mice. Three clusters of transcripts and metabolites involved in energy metabolism, oxidative stress response, and cellular proliferation and differentiation were reduced in CANA-treated male mice. In conclusion, CANA affects bone metabolism mainly via the 'glucose restriction state' it induces and impacts bone cell proliferation and differentiation. These findings underline the effects of SGLT2i on bone health and highlight the need to consider sex-specific responses when developing clinical treatments that alter substrate availability.

Barth syndrome is a mitochondrial disorder with hallmarks of cardiac and skeletal muscle weakness. Barth syndrome is caused by mutation of the X-linked gene Taz, required for cardiolipin remodeling. Previously described germline and conditional Taz knockout models are not ideal for therapeutic development because they lack the combination of robust survival to adulthood, cardiomyopathy, and skeletal muscle weakness. We characterized a cardiac and skeletal muscle-specific Taz knockout model (TazmKO) in which Cre recombinase is expressed from the muscle creatine kinase promoter (mCK-Cre). TazmKO mice survived normally. Cardiolipin composition was abnormal in both heart and skeletal muscle. TazmKO had reduced heart function by 2 months of age, and function progressively declined thereafter. Reduced treadmill endurance and diminished peak oxygen consumption were evident by three months of age, suggesting reduced skeletal muscle function. Electron microscopy showed abnormalities in mitochondrial structure and distribution. Overall, TazmKO mice display diminished cardiac function and exercise capacity while maintaining normal survival. This model will be useful for studying the effects of Taz deficiency in striated muscles and for testing potential therapies for Barth Syndrome.

PURPOSE/OBJECTIVE:To assess the association between neoadjuvant therapy and overall survival (OS) in patients with left-sided resectable pancreatic cancer (RPC) compared to upfront surgery. BACKGROUND:Left-sided pancreatic cancer is associated with worse OS compared to right-sided pancreatic cancer. Although neoadjuvant therapy is currently seen as not effective in patients with RPC, current randomized trials included mostly patients with right-sided RPC. METHODS:International multicenter retrospective study including consecutive patients after left-sided pancreatic resection for pathology-proven RPC, either after neoadjuvant therapy or upfront surgery in 76 centers from 18 countries on 4 continents (2013-2019). Primary endpoint is OS from diagnosis. Time-dependent Cox regression analysis was performed to investigate the association of neoadjuvant therapy with OS, adjusting for confounders at time of diagnosis. Adjusted OS probabilities were calculated. RESULTS:=0.96) involvement. CONCLUSIONS:Neoadjuvant therapy in patients with left-sided RPC was associated with improved OS compared to upfront surgery. The impact of neoadjuvant therapy increased with larger tumor size and higher serum CA19-9 at diagnosis. Randomized controlled trials on neoadjuvant therapy specifically in patients with left-sided RPC are needed.

Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane. We go on to characterize the molecular determinants of substrate specificity using a variety of different substrates as well as protein mutagenesis to probe the binding site. Finally, we present cryogenic electron microscopy structures of Arabidopsis thaliana PIN8 bound to either 2,4-dichlorophenoxyacetic acid or 4-chlorophenoxyacetic acid. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with the transport cycle. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results provide a foundation for future development of novel synthetic auxins and for precision breeding of herbicide-resistant crop plants.

Adipose tissue (AT) is a complex connective tissue with a high relative proportion of adipocytes, which are specialized cells with the ability to store lipids in large droplets. AT is found in multiple discrete depots throughout the body, where it serves as the primary repository for excess calories. In addition, AT has an important role in functions as diverse as insulation, immunity and regulation of metabolic homeostasis. The Human Cell Atlas Adipose Bionetwork was established to support the generation of single-cell atlases of human AT as well as the development of unified approaches and consensus for cell annotation. Here, we provide a first roadmap from this bionetwork, including our suggested cell annotations for humans and mice, with the aim of describing the state of the field and providing guidelines for the production, analysis, interpretation and presentation of AT single-cell data.