FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model.

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homologue ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4.

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.

Stage-based recipient and donor outcome in twin-to-twin transfusion syndrome treated by fetoscopic laser surgery using Solomon technique.

OBJECTIVE: To evaluate twin survival stratified by Quintero stage in patients with twin-to-twin transfusion syndrome (TTTS) after Solomon laser treatment. METHODS: This was a single-center study at Johns Hopkins Center for Fetal Therapy, investigating a cohort of consecutive twin pregnancies treated with the Solomon laser technique for TTTS. Preoperative Quintero stage, perioperative characteristics and obstetric factors were investigated in relation to neonatal survival of the recipient and donor twins at discharge. Determinants of twin survival were evaluated using univariate logistic regression and cumulative survival probability analyses. RESULTS: Of 402 pregnancies with TTTS that underwent Solomon laser treatment, 80 (19.9%) were diagnosed with Quintero Stage-I TTTS, 126 (31.3%) with Stage II, 169 (42.0%) with Stage III and 27 (6.7%) with Stage IV. Post-laser twin anemia polycythemia sequence or recurrent TTTS occurred in 19 (4.7%) patients and 11 (2.7%) required repeat laser surgery. Preterm prelabor rupture of membranes occurred in 150 (37.3%) patients and median gestational age at delivery was 32 + 1 weeks. In 303 (75.4%) patients, both twins were alive at discharge; 67/80 (83.8%) were Stage I, 101/126 (80.2%) were Stage II, 113/169 (66.9%) were Stage III and 22/27 (81.5%) were Stage IV (P = 0.062). Donor twin survival was lower than that of recipients in cases with Stage-III TTTS (118/169 (69.8%) vs 145/169 (85.8%) (χ2 = 26.076, P < 0.0001)). Higher intertwin size discordance and absent or reversed umbilical artery (UA) end-diastolic velocity (EDV) were associated with donor demise (Nagelkerke R2, 0.38; P < 0.001). Overall, spontaneous post-laser donor demise occurred in 53 (39.6%) patients, accounting for the majority of all losses. Cumulative donor survival decreased from 92% to 65% when intertwin size discordance was >30% and to 48% when UA-EDV was absent or reversed (P < 0.001). CONCLUSIONS: The Solomon laser technique achieves TTTS resolution and double twin survival in a high proportion of cases. Recipient and donor survival is comparable unless there is significant intertwin size discordance and placental dysfunction. This degree of unequal placental sharing, typically found in Stage-III TTTS, is the primary factor preventing double survival due to a higher rate of donor demise. © 2024 International Society of Ultrasound in Obstetrics and Gynecology.

The association of microcephaly protein WDR62 with CPAP/IFT88 is required for cilia formation and neocortical development.

WDR62 mutations that result in protein loss, truncation or single amino-acid substitutions are causative for human microcephaly, indicating critical roles in cell expansion required for brain development. WDR62 missense mutations that retain protein expression represent partial loss-of-function mutants that may therefore provide specific insights into radial glial cell processes critical for brain growth. Here we utilized CRISPR/Cas9 approaches to generate three strains of WDR62 mutant mice; WDR62 V66M/V66M and WDR62R439H/R439H mice recapitulate conserved missense mutations found in humans with microcephaly, with the third strain being a null allele (WDR62stop/stop). Each of these mutations resulted in embryonic lethality to varying degrees and gross morphological defects consistent with ciliopathies (dwarfism, anophthalmia and microcephaly). We find that WDR62 mutant proteins (V66M and R439H) localize to the basal body but fail to recruit CPAP. As a consequence, we observe deficient recruitment of IFT88, a protein that is required for cilia formation. This underpins the maintenance of radial glia as WDR62 mutations caused premature differentiation of radial glia resulting in reduced generation of neurons and cortical thinning. These findings highlight the important role of the primary cilium in neocortical expansion and implicate ciliary dysfunction as underlying the pathology of MCPH2 patients.

Healing of sub-critical femoral osteotomies in mice is unaffected by tacrolimus and deletion of recombination activating gene 1.

Clinical management of delayed healing or non-union of long bone fractures and segmental defects poses a substantial orthopaedic challenge. There are suggestions in the literature that bone healing may be enhanced by inhibiting the activities of T and B lymphocytes, but this remains controversial. To examine this matter in more detail, sub-critical-sized segmental defects were created in the femora of mice and it was assessed whether there might be a benefit from the administration of a Food and Drug Administration (FDA)-approved drug that blocks T cell activation (tacrolimus). Defects were stabilised using an internal plate. In certain groups of animals, 1 mg/kg or 10 mg/kg tacrolimus was delivered locally to the defect site for 3 or 7 d using an implanted osmotic pump with a silicon catheter directing drug delivery into the defect area. Healing was monitored by weekly X-ray and assessed at 12 weeks by mechanical testing, µCT and histology. Radiographic and histological evaluations revealed that 100 % of defects healed well regardless of tacrolimus dosage or duration. A comparison of healed C57BL/6 and Rag1-/- femora by µCT and ex vivo torsion testing showed no differences within mouse strains in terms of bone volume, tissue volume, bone volume/tissue volume ratio, shear modulus, torsional rigidity or torsional stiffness. These data failed to support an important role for tacrolimus in modulating the natural healing of segmental defects under those experimental conditions.

Determination of fetal heart rate short-term variation from umbilical artery Doppler waveforms.

OBJECTIVE: To evaluate the feasibility of using umbilical artery (UA) Doppler waveforms to measure fetal heart rate (FHR) short-term variation (STV) across gestation. METHODS: This was a prospective longitudinal study, conducted at two study sites, of 195 pregnancies considered low risk. Pulsed-wave Doppler of the UAs was performed at 4-weekly intervals, between 14 and 40 weeks of gestation, using a standardized imaging protocol. Up to 12 consecutive UA Doppler waveforms were analyzed using offline processing software. FHR STV was calculated using average R-R intervals extracted from the waveforms and baseline corrected for FHR. RESULTS: Baseline-corrected FHR STV increased significantly with gestational age (conditional R2 = 0.37; P < 0.0001) and was correlated inversely with FHR (conditional R2 = 0.54; P < 0.0001). The STV ranged (median (interquartile range)) from 3.5 (2.9-4.1) ms at 14-20 weeks' gestation to 6.3 (4.8-7.7) ms at 34-40 weeks' gestation. The change in heart rate STV did not differ between study sites or individual sonographers. CONCLUSIONS: UA Doppler waveforms offer a robust and feasible method to derive STV of the FHR. It should be emphasized that the UA Doppler-derived STV is not interchangeable with measurements derived with computerized cardiotocography. Accordingly, further investigations are needed to validate associations with outcome, in order to determine the value of concurrent fetal cardiovascular and heart rate evaluations that are possible with the technique described here. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Gs/Gi Regulation of Bone Cell Differentiation: Review and Insights from Engineered Receptors.

G-protein coupled receptors (GPCRs) and their ligands are critical for normal osteoblast formation and function. GPCRs mediate a wide variety of biological processes and are activated by multiple types of extracellular signals, ranging from photons to small molecules to peptides. GPCRs signal through a select number of canonical pathways: the Gs and Gi pathways increase or decrease intracellular cAMP levels, respectively, by acting on adenylate cyclase, while the Gq pathway increases intracellular calcium by activating phospholipase C. In addition, non-canonical GPCR pathways such as ß-arrestin activation are important for osteoblast function. Since many cells express multiple GPCRs, and each individual GPCR may activate multiple signaling pathways, the resulting combinatorial signal provides a mechanism for regulating complex biological processes and effector functions. However, the wide variety of GPCRs, the possibility of multiple receptors acting with signaling redundancy, and the possibility of an individual GPCR activating multiple signaling pathways, also pose challenges for elucidating the role of a particular GPCR. Here, we briefly review the roles of Gs and Gi GPCR signaling in osteoblast function. We describe the successful application of a strategy for directly manipulating the Gs and Gi pathways using engineered receptors. These powerful tools will allow further elucidation of the roles of GPCR signaling in specific lineages of osteoblastic cells, as well as in non-osteoblast cells, all of which remain critical areas of active research.

Vision in Drosophila: seeing the world through a model's eyes.

The fruit fly, Drosophila melanogaster, has been used for decades as a genetic model for unraveling mechanisms of development and behavior. In order to efficiently assign gene functions to cellular and behavioral processes, early measures were often necessarily simple. Much of what is known of developmental pathways was based on disrupting highly regular structures, such as patterns of cells in the eye. Similarly, reliable visual behaviors such as phototaxis and motion responses provided a solid foundation for dissecting vision. Researchers have recently begun to examine how this model organism responds to more complex or naturalistic stimuli by designing novel paradigms that more closely mimic visual behavior in the wild. Alongside these advances, the development of brain-recording strategies allied with novel genetic tools has brought about a new era of Drosophila vision research where neuronal activity can be related to behavior in the natural world.

Drosophila larval motor patterning relies on regulated alternative splicing of Dscam2.

Recent studies capitalizing on the newly complete nanometer-resolution Drosophila larval connectome have made significant advances in identifying the structural basis of motor patterning. However, the molecular mechanisms utilized by neurons to wire these circuits remain poorly understood. In this study we explore how cell-specific expression of two Dscam2 isoforms, which mediate isoform-specific homophilic binding, contributes to motor patterning and output of Drosophila larvae. Ablating Dscam2 isoform diversity resulted in impaired locomotion. Electrophysiological assessment at the neuromuscular junction during fictive locomotion indicated that this behavioral defect was largely caused by weaker bouts of motor neuron activity. Morphological analyses of single motor neurons using MultiColour FlpOut revealed severe errors in dendrite arborization and assessment of cholinergic and GABAergic projections to the motor domain revealed altered morphology of interneuron processes. Loss of Dscam2 did not affect locomotor output, motor neuron activation or dendrite targeting. Our findings thus suggest that locomotor circuit phenotypes arise specifically from inappropriate Dscam2 interactions between premotor interneurons and motor neurons when they express the same isoform. Indeed, we report here that first-order premotor interneurons express Dscam2A. Since motor neurons express Dscam2B, our results provide evidence that Dscam2 isoform expression alternates between synaptic partners in the nerve cord. Our study demonstrates the importance of cell-specific alternative splicing in establishing the circuitry that underlies neuromotor patterning without inducing unwanted intercellular interactions.

Dscam2 mediates axonal tiling in the Drosophila visual system.

Sensory processing centres in both the vertebrate and the invertebrate brain are often organized into reiterated columns, thus facilitating an internal topographic representation of the external world. Cells within each column are arranged in a stereotyped fashion and form precise patterns of synaptic connections within discrete layers. These connections are largely confined to a single column, thereby preserving the spatial information from the periphery. Other neurons integrate this information by connecting to multiple columns. Restricting axons to columns is conceptually similar to tiling. Axons and dendrites of neighbouring neurons of the same class use tiling to form complete, yet non-overlapping, receptive fields. It is thought that, at the molecular level, cell-surface proteins mediate tiling through contact-dependent repulsive interactions, but proteins serving this function have not yet been identified. Here we show that the immunoglobulin superfamily member Dscam2 restricts the connections formed by L1 lamina neurons to columns in the Drosophila visual system. Our data support a model in which Dscam2 homophilic interactions mediate repulsion between neurites of L1 cells in neighbouring columns. We propose that Dscam2 is a tiling receptor for L1 neurons.

Regulated alternative splicing of Dscam2 is required for somatosensory circuit wiring.

Axon and dendrite placement and connectivity is guided by a wide range of secreted and surface molecules in the developing nervous system. Nevertheless, the extraordinary complexity of connections in the brain requires that this repertoire be further diversified to precisely and uniquely regulate cell-cell interactions. One important mechanism for molecular diversification is alternative splicing. Drosophila Down syndrome cell adhesion molecule (Dscam2) undergoes cell type-specific alternative splicing to produce two isoform-specific homophilic binding proteins. Regulated alternative splicing of Dscam2 is important for dendrite and axon patterning, but how this translates to circuit wiring and animal behavior is not well understood. Here, we examined the role of cell-type specific expression of Dscam2 isoforms in regulating synaptic partner selection in the larval somatosensory system. We found that synaptic partners in the nociceptive circuit express different Dscam2 isoforms. Forcing synaptic partners to express a common isoform resulted in nociceptive axon patterning defects and attenuated nocifensive behaviors, indicating that a role for Dscam2 alternative splicing is to ensure that synaptic partners do not express matching isoforms. These results point to a model in which regulated alternative splicing of Dscam2 across populations of neurons restricts connectivity to specific partners and prevents inappropriate synaptic connections.

Intramuscular injection of nerve growth factor as a model of temporomandibular disorder: nature, time-course, and sex differences characterising the pain experience.

Background: Temporomandibular disorder (TMD) is a common condition that frequently transitions to chronic symptoms. Experimental pain models that mimic the symptoms of clinical TMD may be useful in understanding the mechanisms, and sex differences, present in this disorder. Here we aimed to comprehensively characterise the nature and time-course of pain, functional impairment and hyperalgesia induced by repeated intramuscular injection of nerve growth factor (NGF) into the masseter muscle, and to investigate sex differences in the NGF-induced pain experience. Methods: 94 healthy individuals participated in a longitudinal study with 30-day follow-up. NGF was injected into the right masseter muscle on Day 0 and Day 2. Participants attended laboratory sessions to assess pain (Numerical Rating Scale; NRS), functional limitation (mouth opening distance, Jaw Functional Limitation Scale; JFLS) and mechanical sensitization (pressure pain thresholds; PPTs) on Days 0, 2 and 5 and completed twice daily electronic pain dairies from Day 0 to day 30. Results: Peak pain averaged 2.0/10 (95 % CI: 1.6-2.4) at rest and 4.3/10 (95 % CI: 3.9-4.8) on chewing. Pain-free mouth opening distance reduced from 5.0 cm (95 % CI: 4.8-5.1 cm) on Day 0 to 3.7 cm (95 % CI: 3.5-3.9 cm) on Day 5. The greatest reduction in PPTs was observed over the masseter muscle. Females experienced higher pain, greater functional impairment, and greater sensitivity to mechanical stimuli than males. Conclusion: Intramuscular injection of NGF is a useful model with which to explore the mechanisms, and sex differences, present in clinical TMD.

Evaluating Palin Stammering Therapy for School Children (Palin STSC 8-14): protocol for a feasibility randomised controlled trial comparing Palin STSC(8-14) with usual treatment.

BACKGROUND: Having a stammer can have a significant effect on a child's social, emotional and educational development. With approximately 66,000 children in the UK having a stammer, there is a need to establish an adequate evidence base to inform clinical practice. We describe a feasibility trial to explore the effectiveness of a new therapy programme for children aged 8-14: Palin Stammering Therapy for School Children (Palin STSC(8-14)). Preliminary data from the Michael Palin Centre, where the programme was developed, indicate that Palin STSC(8-14) is effective in reducing stammering frequency and impact for children, with beneficial effects for parents too. We will investigate the feasibility of the methods required for a definitive randomised controlled trial to investigate the application of this therapy by NHS speech and language therapists (SLTs), compared with 'treatment as usual' (TAU), beyond the specialist context in which it was developed. METHODS: This is a two-arm feasibility cluster-randomised controlled trial of Palin STSC(8-14) with TAU control arm, and randomisation at the level of the SLT. Quantitative and qualitative data will be collected to examine the following: the recruitment and retention of therapists and families, the acceptability of the research processes and the therapeutic intervention and the appropriateness of the therapy outcome measures. Assessments will be completed by children and parents at baseline and 6 months later, including measures of stammering severity; the impact of child's stammering on both children and parents; child temperament, behaviour and peer relations, anxiety; quality of life; and economic outcomes. There will also be a qualitative process evaluation, including interviews with parents, children, SLTs and SLT managers to explore the acceptability of both the research and therapy methods. Treatment fidelity will be examined through analysis of therapy session records and recordings. DISCUSSION: The findings of this feasibility trial will inform the decision as to whether to progress to a full-scale randomised controlled trial to explore the effectiveness of Palin STSC(8-14) when compared to Treatment as Usual in NHS SLT services. There is a strong need for an evidence-based intervention for school age children who stammer. TRIAL REGISTRATION: ISRCTN. ISRCTN17058884 . Registered on 18 December 2019.

Dscam-mediated repulsion controls tiling and self-avoidance.

Recent studies have uncovered the molecular basis of self-avoidance and tiling, two fundamental principles required for the formation of neural circuits. Both of these wiring strategies are established through homophilic repulsion between Dscam proteins expressed on opposing cell surfaces. In Drosophila, Dscam1 mediates self-avoidance, whereas Dscam2 mediates tiling. By contrast, phenotypes in the retina of the DSCAM mutant mouse indicate that DSCAM functions in both self-avoidance and tiling. These findings suggest that homophilic recognition molecules that have classically been defined as adhesive may also function as repulsive cues and that Dscam proteins specialize in this function.

The Ski proto-oncogene regulates body composition and suppresses lipogenesis.

OBJECTIVE: The Ski gene regulates skeletal muscle differentiation in vitro and and in vivo. In the c-Ski overexpression mouse model there occurs marked skeletal muscle hypertrophy with decreased adipose tissue mass. In this study, we have investigated the underlying molecular mechanisms responsible for the increased skeletal muscle and decreased adipose tissue mass in the c-Ski mouse. APPROACH: Growth and body composition analysis (tissue weights and dual energy X-ray absorptiometry) coupled with skeletal muscle and white adipose gene expression and metabolic phenotyping in c-Ski mice and wild-type (WT) littermate controls was performed. RESULTS: The growth and body composition studies confirmed the early onset of accelerated body growth, with increased lean mass and decreased fat mass in the c-Ski mice. Gene expression analysis in skeletal muscle from c-Ski mice compared with WT mice showed significant differences in myogenic and lipogenic gene expressions that are consistent with the body composition phenotype. Skeletal muscle of c-Ski mice had significantly repressed Smad1, 4, 7 and myostatin gene expression and elevated myogenin, myocyte enhancer factor 2, insulin-like growth factor-1 receptor and insulin-like growth factor-2 expression. Strikingly, expression of the mRNAs encoding the master lipogenic regulators, sterol-regulatory enhancer binding protein 1c (SREBP1c), and the nuclear receptor liver X-receptor-alpha, and their downstream target genes, SCD-1 and FAS, were suppressed in skeletal muscle of c-Ski mice, as were the expressions of other nuclear receptors involved in adipogenesis and metabolism, such as peroxisome proliferator-activated receptor-gamma, glucocorticoid receptor and retinoic acid receptor-related orphan receptor-alpha. Transfection analysis demonstrated Ski repressed the SREBP1c promoter. Moreover, palmitate oxidation and oxidative enzyme activity was increased in skeletal muscle of c-Ski mice. These results suggest that the Ski phenotype involves attenuated lipogenesis, decreased myostatin signalling, coupled to increased myogenesis and fatty acid oxidation. CONCLUSION: Ski regulates several genetic programs and signalling pathways that regulate skeletal muscle and adipose mass to influence body composition development, suggesting that Ski may have a role in risk for obesity and metabolic disease.