Biology subjects

Campeau, P. M.

Publications and source records attributed to Campeau, P. M..

7 recordsLinked to original sources

bioRxiv2025

CHD3 regulates BMP signalling response during cranial neural crest cell specification

CHD3 is a component of the NuRD chromatin remodeling complex. Pathogenic CHD3 variants cause Snijders Blok-Campeau Syndrome, a neurodevelopmental disorder with variable features including developmental delays, intellectual disability, speech/language difficulties, and craniofacial anomalies. To unveil the role of CHD3 in craniofacial development, we differentiated CHD3-KO induced pluripotent stem cells into cranial neural crest cells (CNCCs). CHD3 expression is low in wild-type iPSCs and neuroectoderm, but upregulated during CNCC specification, where it opens the chromatin at BMP-responsive enhancers, to allow binding of DLX5 and other factors. CHD3 loss leads to repression of BMP target genes and an imbalance between BMP and Wnt signalling, ultimately resulting in aberrant mesodermal fate. Consequently, CNCC specification fails, replaced by early-mesoderm identity, which can be partially rescued by titrating Wnt levels. Our findings highlight a novel role for CHD3 as a pivotal regulator of BMP signalling, essential for proper neural crest specification and craniofacial development.

developmental biology↗

bioRxiv2024

TBC1D24 regulates mitochondria and endoplasmic reticulum-mitochondria contact sites

The RabGAP protein TBC1D24 is mutated in several neurological disorders including DOORS syndrome (Deafness, Onycho-Osteodystrophy, Mental Retardation, Seizures). Although TBC1D24 comprises two highly conserved domains -- the Tre2/Bub2/Cdc16 (TBC) domain and the TLDc domain -- both of which seem to play critical roles in cellular functions, its exact physiological function within the cell remains poorly understood. Here, we show that TBC1D24 affects mitochondrial structure and activity. Specifically, both primary fibroblasts from patients with TBC1D24 mutations and cells in which TBC1D24 has been knocked down exhibit fragmented mitochondria, decreased ATP production, and reduced mitochondrial membrane potential. Importantly, loss or mutation of TBC1D24 also alters sites of contact between the endoplasmic reticulum (ER) and mitochondria (ERMCS). These sites are vital for mitochondrial fusion and fission processes, which regulate mitochondrial dynamics and, consequently, mitochondrial activity. Altogether, our results uncover a new role for TBC1D24 in the regulation of mitochondrial functions and ERMCS which likely contribute to the neurological dysfunction present in affected patients.

cell biology↗

bioRxiv2024

Role of Fibronectin in Postnatal Skeletal Development

Fibronectin (FN) is a ubiquitous matrix glycoprotein essential for the physiological development of various tissues. Mutations in FN cause a form of skeletal dysplasia, emphasizing the importance of FN in cartilage and bone development. However, the relevance and functional role of FN during skeletal development remains elusive. We employed conditional knockout mouse models for the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and a double knockout (FNdKO) to determine the relevance of these two principal FN isoforms in postnatal skeletal development spanning from P1 to 2 months of age. We identified a unique topological FN deposition pattern in the mouse limb with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Circulating pFN did not enter the growth plate and was limited to the primary ossification center, whereas cartilage-specific cFN was detected as the major isoform in epiphyseal cartilage. Deletion of either one of the isoforms in single knockouts (cFNKO or pFNKO) only led to subtle changes in some of the analyzed parameters. Complete ablation of both cFN in the growth plate and circulating pFN in plasma resulted in significantly reduced postnatal body weight, body length, and bone length in the FNdKO mice. Assessment of the FNdKO adult bone microarchitecture using micro-CT revealed significantly reduced trabecular bone volume, trabecular network, bone mineral density, and increased bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice showed changes in the proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and type X, reduced apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. FNdKO mice also displayed decreased levels of transforming growth factor-{beta}1 (TGF{beta}1) and downstream phospho-AKT levels, which are critical regulators of chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for proper chondrogenesis and postnatal bone development. Simultaneous deletion of both FN isoforms in the developing cartilage leads to critical TGF{beta}-mediated alterations in chondrogenic differentiation, resulting in bone and skeletal defects. Significance/HighlightsO_LIFN is highly expressed during mouse limb development with increased deposition in resting and hypertrophic chondrocyte zones and the primary ossification center. C_LIO_LICartilage-specific cFN and circulating pFN are distinctly distributed during embryonic and postnatal bone development, with chondrocyte-specific cFN present in the growth plate and pFN limited to trabecular bone and the bone marrow. C_LIO_LIDeletion of both cFN and pFN leads to reduced bone growth during early postnatal development. C_LIO_LIDeletion of cFN and pFN leads to reduced trabecular bone formation, bone mineralization, and increased bone marrow adiposity in 2-month adult mice. C_LIO_LIAbsence of both FN isoforms in the FNdKO mouse model leads to altered chondrogenesis and reduced bone formation. C_LIO_LIFN regulates chondrogenesis via TGF{beta}-mediated phospho-AKT signaling. C_LI

developmental biology↗

bioRxiv2023

Mutations in Fibronectin Dysregulate Chondrogenesis in Corner Fracture Type Spondylometaphyseal Dysplasia

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER and transitioned into lysosomes. Massive amounts of these vesicles were not cleared from the cytosol. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. FN mutant cells also displayed altered FN splice variants under chondrogenic stimuli. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGF{beta}1) expression, crucial for mesenchymal condensation. Exogenous FN or TGF{beta}1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis. Significance /Highlights* SMDCF-causing mutations in fibronectin impair protein secretion in iPSC-derived mesenchymal stem cells. * Mutant fibronectin and ER protein folding chaperones are directly exported from the rough endoplasmic reticulum into vesicles covered with ribosomes, which transition into lysosomes. * The cells cannot clear the massive accumulation of cytosolic vesicles. * Mutations in fibronectin impair stem cell proliferation, mesenchymal condensation, and the differentiation of MSCs into chondrocytes. * Exogenous addition of purified fibronectin or TGF{beta}-1 improves mesenchymal condensation and chondrogenesis of the FN mutant stem cells.

cell biology↗

bioRxiv2020

Ethanolamine phosphate on the second mannose as bridge in GPI anchored proteins: Towards understanding inherited PIGG deficiency

Glycosylphosphatidylinositols (GPIs) are glycolipids that anchor many proteins (GPI-APs) on the cell surface. The core glycan of GPI precursor has three mannoses, which in mammals, are all modified by ethanolamine-phosphate (EthN-P). It is postulated that EthN-P on the third mannose (EthN-P-Man3) is the bridge between GPI to the protein and the second (EthN-P-Man2) is removed after GPI-protein attachment. However, EthN-P-Man2 may not be always transient, as mutations of PIGG, the enzyme that transfers EthN-P to Man2, result in inherited GPI deficiencies (IGDs), characterized by neuronal dysfunctions. Here, we show EthN-P on Man2 is the preferential bridge in some GPI-APs, among them, the ect-5-nucleotidase and netrin G2. We found that CD59, a GPI-AP, is attached via EthN-P-Man2 both in PIGB-knockout cells, in which GPI lacks Man3 and with a small fraction, in wild type cells. Our findings modify the current view of GPI anchoring and provide mechanistic bases of IGDs caused by PIGG mutations.

molecular biology↗

bioRxiv2020

The minor and major spliceosome interact to regulate alternative splicing around minor introns

Mutations in minor spliceosome components are linked to diseases such as Roifman syndrome, Lowry-Wood syndrome, and early-onset cerebellar ataxia (EOCA). Here we report that besides increased minor intron retention, Roifman syndrome and EOCA can also be characterized by elevated alternative splicing (AS) around minor introns. Consistent with the idea that the assembly/activity of the minor spliceosome informs AS in minor intron-containing genes (MIGs), inhibition of all minor spliceosome snRNAs led to upregulated AS. Notably, alternatively spliced MIG isoforms were bound to polysomes in the U11-null dorsal telencephalon, which suggested that aberrant MIG protein expression could contribute to disease pathogenesis. In agreement, expression of an aberrant isoform of the MIG Dctn3 by in utero electroporation, affected radial glial cell divisions. Finally, we show that AS around minor introns is executed by the major spliceosome and is regulated by U11-59K of the minor spliceosome, which forms exon-bridging interactions with proteins of the major spliceosome. Overall, we extend the exon-definition model to MIGs and postulate that disruptions of exon-bridging interactions might contribute to disease severity and pathogenesis.

molecular biology↗

bioRxiv2018

Identification of SMARCD1 as a syndromic intellectual disability gene that is required for memory and context-dependent regulation of neuronal genes in Drosophila

Mutations in several genes encoding components of the SWI/SNF chromatin remodeling complex cause syndromic intellectual disability (ID). Here, we report on 5 individuals with mutations in the SMARCD1 gene, presenting with ID, developmental delay, hypotonia, feeding difficulties, and small extremities. The mutations were proven to be de novo in 4 of the 5 individuals. Mutations in other SWI/SNF components cause Coffin-Siris, Nicolaides-Baraitser, or other syndromic ID disorders. Although the individuals presented here have some clinical overlap with these disorders, they lack the typical facial dysmorphisms. To gain insight into the function of SMARCD1 in neurons, we investigated the Drosophila ortholog, Bap60, in postmitotic memory-forming neurons of the adult Drosophila mushroom body (MB). Targeted knockdown of Bap60 in the MB of adult flies causes defects in long-term memory. Mushroom body specific transcriptome analysis revealed that Bap60 is required for context-dependent expression of genes involved in neuron function and development in juvenile flies when synaptic connections are actively being formed in response to experience. Taken together, we identify SMARCD1 mutations as a novel cause of ID, and establish a role for the SMARCD1 ortholog Bap60 in regulation of neurodevelopmental genes during a critical time window of juvenile adult brain development that is essential in establishing neuronal circuits that are required for learning and memory.

genetics↗