Proteomic Profiling of the First Human Dental Pulp Mesenchymal Stem/Stromal Cells from Carbonic Anhydrase II Deficiency Osteopetrosis Patients

Osteopetrosis is a hereditary disorder characterized by sclerotic, thick, weak, and brittle bone. The biological behavior of mesenchymal cells obtained from osteopetrosis patients has not been well-studied. Isolated mesenchymal stem/stromal cells from dental pulp (DP-MSSCs) of recently extracted deciduous teeth from osteopetrosis (OP) patients and healthy controls (HCs) were compared. We evaluated whether the dental pulp of OP patients has a population of MSSCs with similar multilineage differentiation capability to DP-MSSCs of healthy subjects. Stem/progenitor cells were characterized using immunohistochemistry, flow cytometry, and proteomics. Our DP-MSSCs were strongly positive for CD44, CD73, CD105, and CD90. DP-MSSCs obtained from HC subjects and OP patients showed similar patterns of proliferation and differentiation as well as gene expression. Proteomic analysis identified 1499 unique proteins with 94.3% similarity in global protein fingerprints of HCs and OP patients. Interestingly, we observed subtle differences in expressed proteins of osteopetrosis disease-related in pathways, including MAPK, ERK 1/2, PI3K, and integrin, rather than in the stem cell signaling network. Our findings of similar protein expression signatures in DP-MSSCs of HC and OP patients are of paramount interest, and further in vivo validation study is needed. There is the possibility that OP patients could have their exfoliating deciduous teeth banked for future use in regenerative dentistry.     

Significant Therapeutic Effects of Adult Human Neural Stem Cells for Spinal Cord Injury Are Mediated by Monocyte Chemoattractant Protein-1 (MCP-1)

The limited capability of regeneration in the human central nervous system leads to severe and permanent disabilities following spinal cord injury (SCI) while patients suffer from no viable treatment option. Adult human neural stem cells (ahNSCs) are unique cells derived from the adult human brain, which have the essential characteristics of NSCs. The objective of this study was to characterize the therapeutic effects of ahNSCs isolated from the temporal lobes of focal cortical dysplasia type IIIa for SCI and to elucidate their treatment mechanisms. Results showed that the recovery of motor functions was significantly improved in groups transplanted with ahNSCs, where, in damaged regions of spinal cords, the numbers of both spread and regenerated nerve fibers were observed to be higher than the vehicle group. In addition, the distance between neuronal nuclei in damaged spinal cord tissue was significantly closer in treatment groups than the vehicle group. Based on an immunohistochemistry analysis, those neuroprotective effects of ahNSCs in SCI were found to be mediated by inhibiting apoptosis of spinal cord neurons. Moreover, the analysis of the conditioned medium (CM) of ahNSCs revealed that such neuroprotective effects were mediated by paracrine effects with various types of cytokines released from ahNSCs, where monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) was identified as a key paracrine mediator. These results of ahNSCs could be utilized further in the preclinical and clinical development of effective and safe cell therapeutics for SCI, with no available therapeutic options at present.     

Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

α‐Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α‐galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α‐galactosidase A is known as Fabry disease or Fabry–Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy‐limiting, and eventually life‐threatening complications of ERT. The present study focused on the epitope determination of human α‐galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309–332) recognized by a human monoclonal anti‐αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309–332), was synthesized by solid‐phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K_D=39×10^?9 m ), which is nearly identical to that of the full‐length enzyme (K_D=16×10^?9 m ). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full‐length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.     

Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

Damage induced by oxidative stress is a key driver of the selective motor neuron death in amyotrophic lateral sclerosis (ALS). Mitochondria are among the main producers of ROS, but they also suffer particularly from their harmful effects. Voltage-dependent anion-selective channels (VDACs) are the most represented proteins of the outer mitochondrial membrane where they form pores controlling the permeation of metabolites responsible for mitochondrial functions. For these reasons, VDACs contribute to mitochondrial quality control and the entire energy metabolism of the cell. In this work we assessed in an ALS cell model whether disease-related oxidative stress induces post-translational modifications (PTMs) in VDAC3, a member of the VDAC family of outer mitochondrial membrane channel proteins, known for its role in redox signaling. At this end, protein samples enriched in VDACs were prepared from mitochondria of an ALS model cell line, NSC34 expressing human SOD1G93A, and analyzed by nUHPLC/High-Resolution nESI-MS/MS. Specific over-oxidation, deamidation, succination events were found in VDAC3 from ALS-related NSC34-SOD1G93A but not in non-ALS cell lines. Additionally, we report evidence that some PTMs may affect VDAC3 functionality. In particular, deamidation of Asn215 alone alters single channel behavior in artificial membranes. Overall, our results suggest modifications of VDAC3 that can impact its protective role against ROS, which is particularly important in the ALS context. Data are available via ProteomeXchange with identifier PXD036728.