Faculty Bibliography

Angiopoietin-like 3 (ANGPTL3) inhibits lipases that hydrolyze triglycerides (TGs) in TG-rich lipoproteins (TRLs). We evaluated TRL-TGs, TRL particle (apolipoprotein B), palmitate, and glucose kinetics during a mixed-meal test that included intravenous and oral tracer administrations in people with extremely rare compound heterozygous ANGTPL3 loss-of-function mutations (ANGPTL3^-/- group, n = 3) and matched control participants (n = 7). Multi-organ (liver, muscle, and adipose tissue) insulin sensitivity was evaluated with a two-step hyperinsulinemic-euglycemic clamp procedure and glucose and palmitate tracer infusions. We find that plasma TG and TRL particle concentrations are more than 10-fold lower in the ANGPTL3^-/- than in the control group due to both markedly reduced liver-derived TRL particle and TG secretion rates combined with increased plasma clearance of both liver- and gut-derived TRLs. Palmitate and glucose kinetics during the meal test are not different between the groups. We conclude that the biological function of ANGPTL3 reaches beyond inhibiting intravascular lipase activity.

Huntington's disease (HD) is caused by the expansion of the polyglutamine stretch in huntingtin protein (HTT) resulting in hallmark aggresomes/inclusion bodies (IBs) composed of mutant huntingtin protein (mHTT) and its fragments. Stimulating autophagy to enhance mHTT clearance is considered a potential therapeutic strategy for HD. Our recent evaluation of the autophagic-lysosomal pathway (ALP) in human HD brain reveals upregulated lysosomal biogenesis and relatively normal autophagy flux in early Vonsattel grade brains, but impaired autolysosome clearance in late grade brains, suggesting that autophagy stimulation could have therapeutic benefits as an early clinical intervention. Here, we tested this hypothesis by crossing the Q175 HD knock-in model with our autophagy reporter mouse TRGL (Thy-1-RFP-GFP-LC3) to investigate in vivo neuronal ALP dynamics. In the Q175 and/or TRGL/Q175 mice, mHTT was detected in autophagic vacuoles and also exhibited a high level of colocalization with autophagy receptors p62/SQSTM1 and ubiquitin in the IBs. Compared to the robust lysosomal pathology in late-stage human HD striatum, ALP alterations in Q175 models are also late-onset but milder, that included a lowered phospho-p70S6K level, lysosome depletion, and autolysosome elevation including more poorly acidified autolysosomes and larger-sized lipofuscin granules, reflecting impaired autophagic flux. Administration of a mTOR inhibitor to 6-mo-old TRGL/Q175 normalized lysosome number, ameliorated aggresome pathology while reducing mHTT-, p62-, and ubiquitin-immunoreactivities, suggesting the beneficial potential of autophagy modulation at early stages of disease progression.

BACKGROUND:Plakophilin-2 (PKP2) is a component of the desmosome. Pathogenic variants can lead to arrhythmogenic cardiomyopathy (PKP2-ACM). In PKP2-ACM patients, exercise and catecholamine surges negatively impact arrhythmia incidence and severity. OBJECTIVE:To characterize remodeling of the sympathetic input and adrenergic response in hearts of PKP2-deficient mice (PKP2cKO) subjected to endurance exercise. METHODS:transient dynamics. Separately, we evaluated distribution of sympathetic terminals in PKP2cKO trained hearts vs controls. RESULTS:Exercise led to increased abundance of sarcolemma β1-ARs in control, and decreased abundance in PKP2cKO-myocytes. OCT3 knockdown drastically reduced the response of trained PKP2cKO-myocytes to norepinephrine but not isoproterenol, indicating preserved response to native catecholamines by intracellular (dyad-associated) receptors in the setting of a reduced sarcolemma pool. In tissue, we observed reduced abundance of sympathetic terminals, and heterogeneous distribution across the myocardium. CONCLUSION/CONCLUSIONS:Endurance exercise in PKP2-deficient myocytes leads to reduced pool of functional β1-ARs in the sarcolemma and yet availability of intracellular receptors, which can activate selected (and heterogeneous) routes of intracellular signaling cascades. We speculate that remodeling of nerve terminals affects sympathetic input distribution and hence, regional modulation of excitability and conduction. These changes can facilitate cell-generated triggered activity and heterogeneity of the underlying substrate, setting the stage for life-threatening arrhythmias.

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.

PURPOSE/OBJECTIVE:The purpose of this study is to examine the effect of age on outcomes following repair of acute displaced patella fractures Methods: 248 patients who sustained a displaced patella fracture and underwent open reduction and internal fixation were identified. Patients included underwent a similar operative protocol, were prescribed a standard post-operative protocol of therapy, and were seen at standard follow-up intervals. Patients were divided into groups of < 65 years old (young) and ≥ 65 years old (older). Statistical analysis was run to determine if there was a significant difference in range of knee motion and rate of major complications. RESULTS:Of the 248 patients, 149 were young and 99 were older. The mean age of the older group was 74.5 ± 6.7 and the mean age of the young group was 50 ± 12. Fracture pattern and BMI were similar the groups, however the older group had a higher average CCI (p<0.001). Additionally, the groups had similar length of follow up (p=0.693) and similar mean time to radiographic healing (p=0.533). Older patients had limited knee extension at 6 months (compared young patients (p=0.031). Finally, older patients had a higher rate of all complications compared to young patients. Two percent of older patients developed a fracture related infection (FRI), 4% developed a symptomatic nonunion and 11% were underwent re-operation including removal of hardware, total knee replacement, irrigation and debridement and manipulation under anesthesia. CONCLUSION/CONCLUSIONS:Complication rates following patella fracture fixation in older patients were higher than young patients, despite having similar injury patterns, surgical treatment and follow up. These findings can better inform treating physicians during surgical intervention of older patients with patella fractures.

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.