Stephen J. Warner

MARF and Opa1 Control Mitochondrial and Cardiac Function in Drosophila

Rationale: Mitochondria interact via actions of outer and inner membrane fusion proteins. The role of mitochondrial fusion in functioning of the heart, where mitochondria comprise ≈30% of cardiomyocyte volume and their intermyofilament spatial arrangement with other mitochondria is highly ordered, is unknown. Objective: Model and analyze mitochondrial fusion defects in Drosophila melanogaster heart tubes with tinc&Dgr;4Gal4-directed expression of RNA interference (RNAi) for mitochondrial assembly regulatory factor (MARF) and optic atrophy (Opa)1. Methods and Results: Live imaging analysis revealed that heart tube–specific knockdown of MARF or Opa1 increases mitochondrial morphometric heterogeneity and induces heart tube dilation with profound contractile impairment. Sarcoplasmic reticular structure was unaffected. Cardiomyocyte expression of human mitofusin (mfn)1 or -2 rescued MARF RNAi cardiomyopathy, demonstrating functional homology between Drosophila MARF and human mitofusins. Suppressing mitochondrial fusion increased compensatory expression of nuclear-encoded mitochondrial genes, indicating mitochondrial biogenesis. The MARF RNAi cardiomyopathy was prevented by transgenic expression of superoxide dismutase 1. Conclusions: Mitochondrial fusion is essential to cardiomyocyte mitochondrial function and regeneration. Reactive oxygen species are key mediators of cardiomyopathy in mitochondrial fusion-defective cardiomyocytes. Postulated mitochondrial–endoplasmic reticulum interactions mediated uniquely by mfn2 appear dispensable to functioning of the fly heart.

The Cdc42/Par6/aPKC polarity complex regulates apoptosis-induced compensatory proliferation in epithelia.

BACKGROUND In response to stress- or tissue-damage-induced apoptosis, unaffected epithelial cells undergo compensatory proliferation to maintain the integrity of the epithelium. Proximal signals regulating this response are not fully understood, but c-Jun N-terminal kinase (JNK) activity appears to be critical for both apoptosis and compensatory proliferation. Disruption of epithelial cell apical-basal polarity occurs in early cancer development and is often correlated with increased proliferation by means not fully characterized. We considered whether disruption of the various polarity complexes could provide signals identifying damaged epithelial cells and thus lead to apoptosis-induced compensatory proliferation. RESULTS We identify the Cdc42/Par6/atypical protein kinase C (aPKC) Par polarity complex as uniquely and specifically regulating apoptosis-induced compensatory proliferation in Drosophila epithelia. Genetic depletion of individual components or disruption of formation and localization of this complex, but not other polarity complexes, induces JNK-dependent apoptosis and JNK-dependent compensatory proliferation following radiation injury. When apoptosis execution is blocked, by p35 expression, Cdc42/Par6/aPKC-depleted tissues uniquely hyperproliferate, leading to tissue and organ overgrowth. Disruption of Cdc42/Par6/aPKC leads to activation of JNK through increased Rho1 and Rok activity and Roks capacity to activate myosin but not F-actin. CONCLUSIONS We show that the Cdc42/Par6/aPKC polarity complex influences both a physiologic compensatory proliferation response after irradiation injury and a contrived compensatory non-cell-autonomous hyperproliferation response when cell-autonomous apoptosis, resulting from Cdc42/Par6/aPKC disruption, is inhibited. These results suggest the possibility that in cancer where apoptotic regulation is disrupted, loss of Cdc42/Par6/aPKC polarity complex organization or localization could contribute to tumor hyperproliferation and explain how polarity disruption contributes to tumor development.

Distinct functions for Rho1 in maintaining adherens junctions and apical tension in remodeling epithelia

Maintenance and remodeling of adherens junctions (AJs) and cell shape in epithelia are necessary for the development of functional epithelia and are commonly altered during cancer progression/metastasis. Although formation of nascent AJs has received much attention, whether shared mechanisms are responsible for the maintenance and remodeling of AJs in dynamic epithelia, particularly in vivo, is not clear. Using clonal analysis in the postmitotic Drosophila melanogaster pupal eye epithelium, we demonstrate that Rho1 is required to maintain AJ integrity independent of its role in sustaining apical cell tension. Rho1 depletion in a remodeling postmitotic epithelium disrupts AJs but only when depleted in adjacent cells. Surprisingly, neither of the Rho effectors, Rok or Dia, is necessary downstream of Rho1 to maintain AJs; instead, Rho1 maintains AJs by inhibiting Drosophila epithelial cadherin endocytosis in a Cdc42/Par6-dependent manner. In contrast, depletion of Rho1 in single cells decreases apical tension, and Rok and myosin are necessary, while Dia function also contributes, downstream of Rho1 to sustain apical cell tension.

Differing Src signaling levels have distinct outcomes in Drosophila.

High levels of Src activity are found in a broad spectrum of cancers. The roles of Src and its negative regulator Csk have been extensively studied, although results have often proved contradictory or the relevance to whole organisms is unclear. In Drosophila, overexpression of either Src orthologue resulted in apoptotic cell death, but paradoxically, reducing dCsk activity led to over-proliferation and tissue overgrowth. Here, we show that in Drosophila epithelia in situ, the levels of Src signaling determine the cellular outcome of Src activation. Apoptotic cell death was triggered specifically at high Src signaling levels; lower levels directed antiapoptotic signals while promoting proliferation. Furthermore, our data indicate that expression of kinase-dead Src isoforms do not necessarily act as dominant-negative factors, but can instead increase Src pathway activity, most likely by titrating Csk activity away from endogenous Src. The importance of Src activity levels was emphasized when we examined oncogenic cooperation between Src and Ras: malignant overgrowth was observed specifically when high Src signaling levels were achieved. We propose a model in which low levels of Src signaling promote survival and proliferation during early stages of tumorigenesis, whereas strong Src signaling, coupled with antiapoptotic signals, directs invasive migration and metastasis during advanced tumor stages.

Sport-Specific Outcomes After Anterior Cruciate Ligament Reconstruction

PURPOSE Although anterior cruciate ligament (ACL) reconstruction has been studied extensively in the literature, sport-specific outcomes have not been well-documented. The purpose of this systematic review was to assess sport-specific outcomes after ACL reconstruction in the literature. METHODS We performed a systematic review of the literature to identify studies reporting sport-specific outcomes after primary ACL reconstruction. Included studies were required to have reported standardized outcomes after primary ACL reconstruction for a single sport or comparing between different sports. RESULTS In total 8 studies conformed to all inclusion criteria: 2 Level II studies, 1 Level III study, and 5 Level IV case series. Only 1 study reported comparisons of standardized outcomes between different sports, whereas 7 studies reported standardized outcomes in a single sport. Return to activity was the most common sport-specific outcome reported and varied from 19% (soccer) to 100% (bicycling and rugby), although the methods of measuring this outcome differed. CONCLUSIONS Whereas return to activity after ACL reconstruction appears more likely for bicycling and jogging than for cutting and pivoting sports such as soccer and football, the literature on sport-specific outcomes from ACL reconstruction is limited with minimal data. Further studies are needed to report sport-specific outcomes and return to play after ACL reconstruction. LEVEL OF EVIDENCE Level IV, systematic review of Level II, III, and IV studies.

Cdc42 antagonizes Rho1 activity at adherens junctions to limit epithelial cell apical tension

In epithelia, cells are arranged in an orderly pattern with a defined orientation and shape. Cadherin containing apical adherens junctions (AJs) and the associated actomyosin cytoskeleton likely contribute to epithelial cell shape by providing apical tension. The Rho guanosine triphosphatases are well known regulators of cell junction formation, maintenance, and function. Specifically, Rho promotes actomyosin activity and cell contractility; however, what controls and localizes this Rho activity as epithelia remodel is unresolved. Using mosaic clonal analysis in the Drosophila melanogaster pupal eye, we find that Cdc42 is critical for limiting apical cell tension by antagonizing Rho activity at AJs. Cdc42 localizes Par6-atypical protein kinase C (aPKC) to AJs, where this complex limits Rho1 activity and thus actomyosin contractility, independent of its effects on Wiskott-Aldrich syndrome protein and p21-activated kinase. Thus, in addition to its role in the establishment and maintenance of apical-basal polarity in forming epithelia, the Cdc42-Par6-aPKC polarity complex is required to limit Rho activity at AJs and thus modulate apical tension so as to shape the final epithelium.

Expression of ZIC genes in the development of the chick inner ear and nervous system

ZIC genes, vertebrate homologues of the Drosophila pair‐rule gene odd‐paired (opa), function in embryonic pattern formation, in the early stages of central nervous system neurogenesis and in cerebellar maturation. Mouse Zic genes are expressed in restricted, and in some cases overlapping, patterns during development, particularly in the central and peripheral nervous systems. We identified chick ZIC2 in a differential display analysis of the auditory system designed to find genes up‐regulated after noise trauma. In this study, we examined the expression of chick ZIC1, ZIC2, and ZIC3 by in situ hybridization in normal inner ear development and in the tissues that influence its development, including the hindbrain, the neural crest, and the periotic mesenchyme. Between Hamburger and Hamilton stages 13 and 24, all three ZIC genes were found in the dorsal periotic mesenchyme adjacent to the developing inner ear. ZIC1 mRNA was expressed in the otocyst epithelium between stages 12 and 24, in some sensory tissue, as well as in a striped pattern in the floorplate of the hindbrain that appears to be complementary to that of Chordin, a gene known to regulate ZIC expression in frogs. Chick ZIC genes are also expressed in the neuroectoderm, paraxial mesenchyme, brain, spinal cord, neural crest, and/or the overlying ectoderm as well as the limb buds. In general, ZIC1 and ZIC2 expression patterns overlapped, although ZIC2 expression was less robust; ZIC3 expression was minimal. These observations suggest that ZIC genes, in addition to their known roles in brain development, may play an important role in the development of the chick inner ear. Developmental Dynamics 702–712, 2003.

The Measurement and Clinical Importance of Syndesmotic Reduction After Operative Fixation of Rotational Ankle Fractures

BACKGROUND Rotational ankle fractures often have unstable syndesmotic injuries that require reduction and stabilization. Multiple studies have focused on methods to assess syndesmotic reduction; however, the clinical importance of anatomic syndesmotic reduction remains unclear. The purpose of this study was to determine whether the quality of syndesmotic reduction influenced clinical outcomes following operative treatment of ankle fractures with unstable syndesmotic injuries. METHODS Patients were included from an institutional trauma registry if they had sustained rotational ankle fractures with intraoperative evidence of syndesmotic instability requiring syndesmotic reduction and stabilization. Patients with at least twelve months of disease-specific, patient-reported clinical outcomes were included. Computed tomography (CT) imaging of both ankles was performed within two days postoperatively for all patients. Four previously utilized methods of assessing syndesmotic reduction using axial CT images of the operatively treated and the contralateral ankle were used. RESULTS A total of 155 patients met the study inclusion criteria and underwent analysis. The four methods used to assess syndesmotic reduction had reliabilities ranging from moderate to almost perfect (intraclass correlation coefficient [2,1] range = 0.544 to 0.821). Measurements of the uninjured syndesmosis were consistent with those in several previous studies of normal syndesmotic morphology, and the four methods of syndesmotic assessment had strong internal consistency. The mean measurement differences between the injured and normal ankles ranged from 1.32 to 1.88 mm of displacement and averaged 5.75° of rotation. There were no correlations noted between any of the four syndesmotic reduction assessment methods and any Foot and Ankle Outcome Score domains. CONCLUSIONS Within the range of syndesmotic malreductions studied, the quality of syndesmotic reduction did not significantly influence clinical outcomes. These results challenge previous definitions of syndesmotic malreduction and the clinical importance of minor syndesmotic changes. It remains unclear, however, whether greater magnitudes of syndesmotic malreduction than those seen in this cohort would lead to inferior patient-reported outcomes.

Correlation Between the Lauge-Hansen Classification and Ligament Injuries in Ankle Fractures.

Objective: To evaluate the ability of the Lauge-Hansen classification to predict ligament injuries in ankle fractures using magnetic resonance imaging (MRI) and intraoperative findings. Design: Prospective evaluation in consecutive patients. Setting: Academic level 1 trauma center. Patients: Three-hundred patients with an operatively treated ankle fracture who met the inclusion and exclusion criteria. Intervention: Injury ankle radiographs were assigned to a Lauge-Hansen classification. MRI scans were obtained to evaluate the syndesmotic and deltoid ligaments. A Lauge-Hansen classification for each patient was recorded based on intraoperative findings. Main Outcome Measurements: Comparisons were made between the predicted ankle ligamentous injuries based on radiographic Lauge-Hansen classifications, preoperative MRI analyses, and intraoperative findings. Results: On the basis of the Lauge-Hansen system and injury radiographs, 77% (231/300) were classified as supination external rotation, 13% (40/300) were pronation external rotation, 4% (11/300) were supination adduction, <1% (1/300) was pronation abduction, and 6% (17/300) were not classifiable. Of the 283 fractures that were classified into Lauge-Hansen classes, 266 (94%) had MRI readings of ligamentous injuries consistent with the Lauge-Hansen predictions. Intraoperative findings also highly correlated with the Lauge-Hansen class of ankle fractures, with nearly complete agreement. Comparing MRI and intraoperative findings revealed discrepancies in 6% (16/283) of ankle fracture classifications. Conclusions: In our large cohort of patients, comparisons between injury radiographs, preoperative MRI, and intraoperative findings suggest that the Lauge-Hansen system is an accurate predictor of ligamentous injuries. The predictions based on the Lauge-Hansen system can be useful for fracture reduction maneuvers and operative planning. Level of Evidence: Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Multiplanar Fixation for Patella Fractures Using a Low-Profile Mesh Plate.

Summary: Patella fractures are challenging orthopaedic injuries. Many commonly used fixation techniques can be ineffective and lead to poor clinical outcomes even with satisfactory reductions and fracture healing. In this investigation, we present the technique of cage plate fixation of patella fractures and the clinical outcomes of 9 initial patients surgically treated at our institution. This technique allows direct visualization of the articular reduction, provides multiplanar fixation, effectively stabilizes inferior pole comminution, and reduces the risk of patella vascular disruption. Using this technique, we have achieved excellent functional and radiographic outcomes.