Phenotypic and molecular characterization of human SPG10 model in Drosophila melanogaster and its link to BMP signaling

Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous neurodegenerative disorders characterized by progressive spasticity of lower extremities as a consequence of axonopathy of the longest cortico spinal tract. Defects in axonal transport, ER membrane modeling, mitochondrial function, DNA repair, autophagy, lipid metabolism and myelination have been linked to HSP. SPG10, a subtype of HSP is caused due to mutations in the anterograde microtubular motor protein kinesin. Kinesin mutation impairs axonal transport leading to synaptic dysfunction. My study was aimed at the phenotypic and molecular characterization of the human SPG10 model in Drosophila melanogaster and to investigate its suspected interplay with BMP signaling. The mutation in kinesin protein in human SPG10 has been mapped to position N256S. This point mutation was inserted into Drosphila kinesin heavy chain (Khc) at the N262S position and the mutated kinesin protein was ectopically expressed using tissue specific drivers. The Drosophila KhcN262S mutants were characterized by reduced survival, behavioral impairments, axonal swellings, synaptic protein depletion at distal NMJs, cytoskeletal disability, developmental delay, and synaptic degeneration. Ectopic overexpression of wild type kinesin alongside mutated kinesin (KhcN262S+wt) partially rescued the HSP pathology thus revealing the dominant negative nature of this mutation. KhcN262S mutation affected both anterograde and retrograde transport in Drosophila larvae. Impairment of long distance transport within axons directly contributes to synaptic defects by perturbing the BMP signaling pathway which is essential for synapse maintenance and function. Till date a common pathological mechanism in HSPs remains to be identified. Atleast 4 human SPGs have been linked to altered BMP signaling. The key pathological features of downregulated BMP signaling in Drosophila resemble that of the KhcN262S Drosophila model. A previous study in the lab show reduced pMad (transcription factor of BMP target gene, Trio) levels in KhcN262S larval motor neuron cell bodies. Assuming that the phenotypical defects in the KhcN262S Drosophila model may be due to altered BMP signaling, we hypothesized that upregulation of BMP signaling in KhcN262S mutants could rescue the synaptic and behavioral defects via cytoskeletal stabilization. However, neither the overexpression of constitutively active BMP receptor Tkv-CA nor the overexpression of target gene Rac-GEF Trio was able to rescue the KhcN26S pathology significantly. In- vivo study using YFP tagged Tkv showed impairments in retrograde transport of Tkv which was consistent with reduced pMad levels observed in motor neuron cell bodies of KhcN262S+TkV-CA larvae. Though overexpression of Trio in mutants partially rescued the cargo trafficking it did not suffice synaptic or behavioral rescue owing to its tight regulation and influence on numerous other genes. Since Trio is the only BMP target gene known, it does not rule out regulation of other unidentified genes. Finally, molecular mechanism involved in KhcN262S induced SPG10 pathology is quite complex since both anterograde and retrograde transport is impaired, hence BMP signaling could only be one among the many signaling pathways affected, and sole upregulation of which alone does not suffice rescue. My study has successfully characterized the severity of the SPG10 model in Drosophila at the molecular level as well as behavioral level. After testing our hypothesis, arising from similarity of phenotypes and previously described interplay at molecular level, a connection between SPG10 pathology and BMP signaling seems highly plausible, yet our proposed mechanism of rescue was only partially successful

Characterisation of porous knitted titanium for replacement of intervertebral disc nucleus pulposus

Effective restoration of human intervertebral disc degeneration is challenged by numerous limitations of the currently available spinal fusion and arthroplasty treatment strategies. Consequently, use of artificial biomaterial implant is gaining attention as a potential therapeutic strategy. Our study is aimed at investigating and characterizing a novel knitted titanium (Ti6Al4V) implant for the replacement of nucleus pulposus to treat early stages of chronic intervertebral disc degeneration. Specific knitted geometry of the scaffold with a porosity of 67.67 ± 0.824% was used to overcome tissue integration failures. Furthermore, to improve the wear resistance without impairing original mechanical strength, electro-polishing step was employed. Electro-polishing treatment changed a surface roughness from 15.22 ± 3.28 to 4.35 ± 0.87 μm without affecting its wettability which remained at 81.03 ± 8.5°. Subsequently, cellular responses of human mesenchymal stem cells (SCP1 cell line) and human primary chondrocytes were investigated which showed positive responses in terms of adherence and viability. Surface wettability was further enhanced to super hydrophilic nature by oxygen plasma treatment, which eventually caused substantial increase in the proliferation of SCP1 cells and primary chondrocytes. Our study implies that owing to scaffolds physicochemical and biocompatible properties, it could improve the clinical performance of nucleus pulposus replacement

Nicotine and Cotinine Inhibit Catalase and Glutathione Reductase Activity Contributing to the Impaired Osteogenesis of SCP-1 Cells Exposed to Cigarette Smoke

Cigarette smoking has been identified as a major risk factor for osteoporosis decades ago. Several studies have shown a direct relationship between cigarette smoking, decreased bone mineral density, and impaired fracture healing. However, the mechanisms behind impaired fracture healing and cigarette smoking are yet to be elucidated. Migration and osteogenesis of mesenchymal stem/stromal cells (MSCs) into the fracture site play a vital role in the process of fracture healing. In human nicotine, the most pharmacologically active and major addictive component present in tobacco gets rapidly metabolized to the more stable cotinine. This study demonstrates that physiological concentrations of both nicotine and cotinine do not affect the osteogenic differentiation of MSCs. However, cigarette smoke exposure induces oxidative stress by increasing superoxide radicals and reducing intracellular glutathione in MSCs, negatively affecting osteogenic differentiation. Although, not actively producing reactive oxygen species (ROS) nicotine and cotinine inhibit catalase and glutathione reductase activity, contributing to an accumulation of ROS by cigarette smoke exposure. Coincubation with N-acetylcysteine or L-ascorbate improves impaired osteogenesis caused by cigarette smoke exposure by both activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and scavenging of ROS, which thus might represent therapeutic targets to support fracture healing in smokers

Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs—The Potential Role in Increasing Anabolic Response

Musculoskeletal disorders, such as osteoarthritis and intervertebral disc degeneration are causes of morbidity, which concomitantly burdens the health and social care systems worldwide, with massive costs. Link N peptide has recently been described as a novel anabolic stimulator for intervertebral disc repair. In this study, we analyzed the influence on anabolic response, by delivering synthetic Link N encoding mRNA into primary human chondrocytes and mesenchymal stromal cells (SCP1 cells). Furthermore, both cell types were seeded on knitted titanium scaffolds, and the influence of Link N peptide mRNA for possible tissue engineering applications was investigated. Synthetic modified Link N mRNA was efficiently delivered into both cell types and cell transfection resulted in an enhanced expression of aggrecan, Sox 9, and type II collagen with a decreased expression of type X collagen. Interestingly, despite increased expression of BMP2 and BMP7, BMP signaling was repressed and TGFβ signaling was boosted by Link N transfection in mesenchymal stromal cells, suggesting possible regulatory mechanisms. Thus, the exogenous delivery of Link N peptide mRNA into cells augmented an anabolic response and thereby increased extracellular matrix synthesis. Considering these findings, we suppose that the cultivation of cells on knitted titanium scaffolds and the exogenous delivery of Link N peptide mRNA into cells could mechanically support the stability of tissue-engineered constructs and improve the synthesis of extracellular matrix by seeded cells. This method can provide a potent strategy for articular cartilage and intervertebral disc regeneration

Knockdown of <i>Hsc70-5/mortalin</i> Induces Loss of Synaptic Mitochondria in a <i>Drosophila</i> Parkinson’s Disease Model

<div><p>Mortalin is an essential component of the molecular machinery that imports nuclear-encoded proteins into mitochondria, assists in their folding, and protects against damage upon accumulation of dysfunctional, unfolded proteins in aging mitochondria. Mortalin dysfunction associated with Parkinson’s disease (PD) increases the vulnerability of cultured cells to proteolytic stress and leads to changes in mitochondrial function and morphology. To date, <i>Drosophila melanogaster</i> has been successfully used to investigate pathogenesis following the loss of several other PD-associated genes. We generated the first loss-of-<i>Hsc70-5/mortalin</i>-function <i>Drosophila</i> model. The reduction of Mortalin expression recapitulates some of the defects observed in the existing <i>Drosophila</i> PD-models, which include reduced ATP levels, abnormal wing posture, shortened life span, and reduced spontaneous locomotor and climbing ability. Dopaminergic neurons seem to be more sensitive to the loss of <i>mortalin</i> than other neuronal sub-types and non-neuronal tissues. The loss of synaptic mitochondria is an early pathological change that might cause later degenerative events. It precedes both behavioral abnormalities and structural changes at the neuromuscular junction (NMJ) of <i>mortalin</i>-knockdown larvae that exhibit increased mitochondrial fragmentation. Autophagy is concomitantly up-regulated, suggesting that mitochondria are degraded via mitophagy. <i>Ex vivo</i> data from human fibroblasts identifies increased mitophagy as an early pathological change that precedes apoptosis. Given the specificity of the observed defects, we are confident that the loss-of-mortalin model presented in this study will be useful for further dissection of the complex network of pathways that underlie the development of mitochondrial parkinsonism.</p></div

Pan-neuronal knockdown of <i>mortalin</i> induced autophagy at the larval NMJ.

<p>(<b>A</b>) <i>Drosophila</i> VNCs of control (elav<i>>white^RNAi</i>) and elav<i>>mort^GD</i> larvae labeled with the autophagosomal ATG8-mRFP marker. No obvious change in the ATG8-mRFP signal was detected upon <i>mortalin</i> knockdown. Gamma values were adjusted to 0.75 Scale bar: 50 µm. (<b>B</b>) Autophagosomes were detected as the strong accumulation of ATG8-mRFP signal at the <i>Drosophila</i> NMJ. The false color look-up table “Green-Fire-Blue” allows the separation of autophagosomes from the diffuse ATG8-mRFP signal. Scale bar: 10 µm. (<b>C</b>) Confocal images of synaptic boutons at NMJ 4 in control (elav<i>>white^RNAi</i>) and elav<i>>mort^GD</i> larvae. Neuronal membranes and autophagosomes are shown in green and magenta, respectively. Scale bar: 5 µm. (<b>D, E</b>) Statistical analysis revealed increases in ATG8-mRFP puncta abundance (<b>D</b>) and size (<b>E</b>). Statistical significance was determined by using an unpaired, two-tailed Student’s t-test.</p