Biology subjects

Ghanem, S.

Publications and source records attributed to Ghanem, S..

3 recordsLinked to original sources

bioRxiv2024

Successful delivery of CRISPR-Cas9 with a baculovirus vector for insect brain targets

CRISPR-Cas9 (clustered, regularly interspaced, short palindromic repeats with CRISPR-associated protein 9) is a powerful, versatile, and cost-effective molecular tool that can be used for genetic engineering purposes and beyond1 and is especially suited for non-model organisms2. Effective delivery of this system, however, remains a challenge for in vivo genetic manipulation of specific tissues3, particularly the brain4, and in adult indivuduals5,6. We designed a new CRISPR-Cas9 plasmid that was inserted into a baculovirus vector to knockdown the octopamine beta subtype 2 receptor (AmOct{beta}2R), a transmembrane protein found in the mushroom body neurons of the honey bee (Apis mellifera) brain, to determine if octopamine plays a role in appetite regulation. We first confirmed that gene editing of AmOct{beta}2R is possible with Sanger sequencing. We then demonstrated expression of the CRISPR-Cas9 system with the baculovirus vector in vitro using live cell imaging, flow cytometry analysis, and in vivo using confocal imaging, showing widespread expression in the cells and throughout the honey bee brain, three days post treatment. There was also in vitro and in vivo knockdown of AmOct{beta}2R three days post-infection, that corresponded with appetite suppression in starved forager bees. Our findings suggest that we successfully delivered the CRISPR-Cas9 system and knocked down AmOctB2R in neuronal cells of the honey bee brain that were previously inaccessible due to the blood brain barrier and lack of infectivity of lentivirus vectors7. The newly characterized AmOct{beta}2R8 can now be assigned a functional role and other targets for gene editing are now possible using this CRISPR-Cas9 system. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/624635v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@9fba0eorg.highwire.dtl.DTLVardef@7025dforg.highwire.dtl.DTLVardef@c6706borg.highwire.dtl.DTLVardef@1fea727_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Seed-competent alpha-synuclein pathology in metachromatic leukodystrophy: the expanding spectrum of alpha-synucleinopathy in sphingolipidoses

Metachromatic leukodystrophy (MLD) is a rare - typically paediatric - sphingolipid storage disorder resulting from bi-allelic pathogenic variants in the ARSA gene, encoding the lysosomal arylsulphatase A (ASA). Heterozygous variants in ARSA are associated with risk of Lewy body diseases (LBD), a group of age-associated neurodegenerative disorders characterised by the accumulation of the protein -synuclein; however, no study has yet determined whether -synuclein with putative pathological features is observed in MLD brain tissue. We examined post-mortem brain tissue from MLD cases (N=5, age 2-33) compared to matched control cases using histological approaches and -synuclein seeding amplification assay (SAA). Juvenile-onset MLD cases exhibited granular -synuclein deposits in neurons of regions prone to neuronal pathology in MLD, and seed-competent conformers that generated atypical short, twisted fibrils on SAA. In contrast, infantile-onset MLD cases gave only variably positive reactions on SAA. In summary, this study suggests MLD cases manifest -synuclein pathology reminiscent of that observed in LBD, even in juvenile populations, further expanding the spectrum of sphingolipid storage disorders associated with the aggregation of -synuclein. These findings have important implications for understanding the disease process of both LBD and MLD, potentially highlighting novel pathways for therapeutic interventions in both conditions.

neuroscience↗

bioRxiv2024

TGF-β Signaling in Cranial Neural Crest Affects Late-Stage Mandibular Bone Resorption and Length

Malocclusions are common craniofacial malformations which cause quality of life and health problems if left untreated. Unfortunately, the current treatment for severe skeletal malocclusion is invasive surgery. Developing improved therapeutic options requires a deeper understanding of the cellular mechanisms responsible for determining jaw bone length. We have recently shown that neural crest mesenchyme (NCM) can alter jaw length by controlling recruitment and function of mesoderm-derived osteoclasts. Transforming growth factor beta (TGF-{beta}) signaling is critical to craniofacial development by directing bone resorption and formation, and heterozygous mutations in TGF-{beta} type I receptor (TGFBR1) are associated with micrognathia in humans. To identify what role TGF-{beta} signaling in NCM plays in controlling osteoclasts during mandibular development, mandibles of mouse embryos deficient in the gene encoding Tgfbr1 specifically in NCM were analyzed. Our lab and others have demonstrated that Tgfbr1fl/fl;Wnt1-Cre mice display significantly shorter mandibles with no condylar, coronoid, or angular processes. We hypothesize that TGF-{beta} signaling in NCM can also direct later bone remodeling and further regulate late embryonic jaw bone length. Interestingly, analysis of mandibular bone through micro-computed tomography and Massons trichrome revealed no significant difference in bone quality between the Tgfbr1fl/fl;Wnt1-Cre mice and controls, as measured by bone perimeter/bone area, trabecular rod-like diameter, number and separation, and gene expression of Collagen type 1 alpha 1 (Col11) and Matrix metalloproteinase 13 (Mmp13). Though there was not a difference in localization of bone resorption within the mandible indicated by TRAP staining, Tgfbr1fl/fl;Wnt1-Cre mice had approximately three-fold less osteoclast number and perimeter than controls. Gene expression of receptor activator of nuclear factor kappa-{beta} (Rank) and Mmp9, markers of osteoclasts and their activity, also showed a three-fold decrease in Tgfbr1fl/fl;Wnt1-Cre mandibles. Evaluation of osteoblast-to-osteoclast signaling revealed no significant difference between Tgfbr1fl/fl;Wnt1-Cre mandibles and controls, leaving the specific mechanism unresolved. Finally, pharmacological inhibition of Tgfbr1 signaling during the initiation of bone mineralization and resorption significantly shortened jaw length in embryos. We conclude that TGF-{beta} signaling in NCM decreases mesoderm-derived osteoclast number, that TGF-{beta} signaling in NCM impacts jaw length late in development, and that this osteoblast-to-osteoclast communication may be occurring through an undescribed mechanism.

developmental biology↗