Faculty Bibliography

Individuals with Down syndrome (DS) produce less saliva for unknown reasons resulting in chronic periodontal disease with systemic detrimental effects. Using the (Dp(16)1Yey) mouse model of DS we define the molecular mechanisms of hyposalivation and potential links to periodontal disease. We show that Dp(16)1Yey mice produce less saliva and have a higher immune burden in the salivary glands. We demonstrate that store operated calcium entry (SOCE), required for saliva secretion, is deficient in the salivary glands of Dp(16)1Yey mice. SOCE is also reduced in iPSCs from an individual with DS. We show that the oral and gut microbiomes of Dp(16)1Yey mice have abundant succinate-associated microbes and high succinate levels in the serum. We highlight associations between altered Ca²⁺ handling and hyposalivation, dysbiosis, and periodontal disease in DS. The administration of pilocarpine in Dp(16)1Yey mice increased salivation, suggesting that cholinergic agonists might be useful to improve the oral health of individual with DS.

Ameloblasts are specialized epithelial cells that form enamel during the secretory and maturation stages, the latter involving an increase in Ca²⁺ transport to mineralize the enamel crystals. During enamel formation, ameloblasts travel several microns while secreting a matrix and are surrounded by several cell layers in the confined space of the enamel organ. Presumably, ameloblasts are subjected to mechanical stimuli e.g. pressure, stretch. Mechanosensitive (MS) or stretch-gated channels are expressed in the membranes of many cells including mineralizing cells. The opening of MS channels occurs in response to physical stimuli and results in the influx of ions. Piezo1 is a non-selective class of MS channel permeable to Ca²⁺ and hence it may contribute to Ca²⁺ homeostasis in ameloblasts. Here we show that secretory and maturation stage ameloblasts express similar protein levels of Piezo1. Cultured rat primary secretory and maturation stage ameloblasts showed stretch-activated currents by patch-clamp. Ameloblasts loaded with the cytosolic Ca²⁺ indicator Fura-2 were also stimulated with the Piezo1-selective activator Yoda1. We show that ameloblasts are sensitive to Piezo1 stimulation which evoked an increase in cytosolic Ca²⁺. This effect was inhibited by Piezo1 blockers. Mechanical analysis of the incisors of Piezo1 cKO mice showed no alterations in hardness or elastic modulus relative to littermate control mice. Our work provides the first evidence that Piezo1 channels are functional in both ameloblast stages and their activation leads to an elevation in cytosolic Ca²⁺, however, Piezo1 does not appear to be essential for enamel mineralization.

A key aspect of biological studies is the reliance on computation, including the use of mathematical models to mimic the behaviour of cellular functions and networks. Quantitative descriptions of biological processes typically rely on physics and chemistry and thus the generated models are mechanistic e.g. they explain behavior. With some exceptions, such models have been lacking in studies of ameloblasts biology and physiology, in part due to the absence of experimental observations. Here we provide a computational model for Ca²⁺ dynamics in ameloblasts based on recent quantitative data, allowing us to investigate how dynamic forces operate in ameloblasts at the secretory and maturation stages. We simulate the effects of mutations occurring in key Ca²⁺ handling proteins. Finally, we compare how ameloblasts differ from other non-mineralizing cell systems.

The K⁺-dependent Na⁺/Ca²⁺-exchanger (NCKX) family comprises five members (NCKX1-5) that serve as key regulators of Ca²⁺ homeostasis. The fourth member, NCKX4, has been implicated in several neuronal processes, including vision, olfaction, and hypothalamic satiety signaling. We recently showed high NCKX4 expression in the mossy fibers of the hippocampus, a brain region essential for learning and memory. In this study, we investigated the impact of NCKX4 loss on Ca²⁺ dynamics in primary hippocampal neurons and assessed hippocampal-dependent behaviour in Nckx4^-/- mice. Using Ca²⁺ imaging, we found that NCKX4 actively contributes to Ca²⁺ homeostasis in hippocampal cells and its loss is not compensated for by other Ca²⁺ transporters. Neurons lacking NCKX4 had significantly reduced rates of total Na⁺/Ca²⁺-exchange activity and showed a decrease in the rate of Ca²⁺ clearance following a depolarization event. Complementary behavioural testing showed deficits in contextual memory formation and recall, as well as reduced anxiety when placed in an open field, in Nckx4^-/- mice. These findings reveal a previously unrecognized role for NCKX4 in regulating hippocampal Ca²⁺ dynamics, and support the hypothesis that NCKX4 expression is mechanistically linked to learning and memory.

Ageing is the primary non-modifiable risk factor for cardiovascular diseases (CVDs), with older women facing a greater risk of CVDs than age-matched men. Vascular smooth muscle cells (VSMCs) dysfunction and impaired calcium (Ca²⁺) handling are recognized as central contributors to arterial stiffening and vascular calcification. However, the molecular and functional determinants of Ca²⁺ clearance that drive vascular ageing remain poorly understood. We identify the (Na⁺)-sodium/Ca²⁺-calcium (K⁺)-potassium-dependent exchanger 4 (NCKX4) as a critical functional regulator of VSMC Ca²⁺ clearance and vascular integrity. Expression of NCKX4 (encoded by Slc24A4) was significantly reduced in aortae from aged (72-78 weeks) mice. Aged-related loss of NCKX4 impaired Ca²⁺ clearance function and increased Ca²⁺-phosphate mineralization. Notably, young (12-15 weeks) Nckx4^-/- mice exhibited elastic fibre fragmentation, collagen accumulation, arterial wall thickening and extracellular matrix (ECM) remodelling, which are hallmarks of vascular ageing that closely resembled those observed in aged wild-type mice. Transcriptomic profiling revealed that loss of NCKX4 alters pathways associated with Ca²⁺-integrin signalling, ECM turnover and mineralization, including dysregulation of anchorage integrins, microfibril-stabilizing components, osteogenic drivers and profibrotic integrins. Collectively, these findings demonstrate that impaired Ca²⁺ clearance promotes maladaptive inside-out integrin signalling, disrupting VSMC anchorage, ECM homeostasis and mineralization. Our results establish NCKX4 as a previously unrecognized determinant of vascular ageing, and its decline accelerates premature arterial remodelling and calcification. This study positions NCKX4 as a mechanistic link between age-dependent vascular vulnerability and arterial stiffening, with implications for novel therapeutic strategies targeting Ca²⁺ handling to prevent CVDs. KEY POINTS: Dysfunctional calcium (Ca²⁺) handling in vascular smooth muscle cells (VSMCs) increases susceptibility to cardiovascular diseases (CVDs), vascular calcification, stiffness and aortic remodelling. The K⁺-dependent Na⁺/Ca²⁺ exchanger NCKX4 mediates high-capacity Ca²⁺ clearance extrusion, thereby maintaining VSMC Ca^{2 +} homeostasis. NCKX4 (encoded by Slc24a4) expression markedly declines in the aorta and VSMCs of aged (72-78 weeks) mice. Genetic deletion or age-related loss of NCKX4 impairs Ca²⁺ clearance, leading to enhanced VSMCs calcification, premature arterial remodelling through disruption of Ca²⁺-mediated integrin-ECM signalling. NCKX4 is a newly identified mechanistic driver of vascular ageing and a potential therapeutic target for early detection, preservation of vascular integrity and mitigation of age-related CVDs risk.

Dental enamel protects against the invasion of bacterial pathogens deep into the innervated layers of the tooth. Hereditary enamel disorders referred to as amelogenesis imperfecta (AI) can severely affect the development and mineralization of dental enamel compromising these functions. This rare disorder is often visible, carries a significant psychological and financial burden, and cosegregates with disease in other organs. Pathological variants in over 100 genes affect the enamel formation. Here, we describe the biology of enamel formation focusing on pathogenic variants underlying AI. We provide a computational model encapsulating new advances in calcium regulation during enamel formation. We also describe the psychological and financial burden of AI, its impact in systemic health, and discuss recent developments in diagnostic panels to detect AI.

Sjogren's Disease (SjD) is an autoimmune disorder characterized by salivary and lacrimal gland dysfunction and immune cell infiltration leading to gland inflammation and destruction. Although SjD is a common disease, its pathogenesis is not fully understood. In this study, we conducted a single-cell transcriptome analysis of peripheral blood mononuclear cells (PBMC) from patients with SjD and symptomatic non-SjD controls to identify cell types and functional changes involved in SjD pathogenesis. All PBMCs populations showed marked differences in gene expression between SjD patients and controls, particularly an increase in interferon (IFN) signaling gene signatures. T and B cells of SjD patients displayed a depletion of ribosomal gene expression and pathways linked to protein translation. SjD patients had increased frequencies of naive B cells, which featured a unique gene expression profile (GEP) distinct from controls and had hallmarks of B cell hyperactivation. Non-negative matrix factorization (NMF) also identified several non-overlapping GEPs in CD4⁺ and CD8⁺ T cells with differential usage in SjD patients and controls. Of these, only the Th1 activation GEP was enriched in T cells of SjD patients whereas the other two GEPs were depleted in T cells, emphasizing the important role of Th1 cells in SjD. Our study provides evidence for aberrant and unique gene expression patterns in both B and T lymphocytes of SjD patients that point to their altered activation states and may provide new insights into the pathogenesis of SjD.

OBJECTIVE:This study follows up on our recent morphological analysis of the juvenile maxilla from Mugharet el'Aliya, Morocco. Although this specimen shows a reportedly archaic morphology, likely due to its large size, 3D shape analyses indicated affinities with early Homo sapiens. Here, we conducted an in-depth comparative investigation of the associated dentition to further clarify this individual's phylogenetic and taxonomic affinities. MATERIALS AND METHODS/METHODS:Our analyses were based on three kinds of data: (a) external crown dimensions and non-metric features, analyzed via summary statistics; (b) CT scan data enabling the study of internal structures (enamel-dentine junction) via geometric morphometrics; and (c) high-resolution replicas of the external surface of the upper canine enabling the study of perikymata numbers via probability functions. The comparative samples included Middle Pleistocene (Chibanian) Europeans and Africans, Neanderthals, and early and later H. sapiens. RESULTS:Mugharet el'Aliya showed the greatest similarities in external and internal tooth morphology with early and later H. sapiens. Perikymata counts cluster the upper canine with H. sapiens. However, its canine and fourth premolar are megadont at a level generally atypical for H. sapiens. DISCUSSION/CONCLUSIONS:Our analyses of the dentition of the Mugharet el'Aliya individual support our previous findings on the morphological analysis of the maxilla, placing this fossil closest to H. sapiens. Our study further strengthens the evidence connecting fossils from the North African Aterian to those from Western Asia, especially Qafzeh. We also provide the first comparative analysis of a permanent upper canine from the Aterian fossil record.

Ca2+ signaling via the store operated Ca2+ entry (SOCE) mediated by STIM1 and STIM2 proteins and the ORAI1 Ca2+ channel is important in saliva fluid secretion and has been associated with Sjogren's disease (SjD). However, there are no studies addressing STIM1/2 dysfunction in salivary glands or SjD in animal models. We report that mice lacking Stim1 and Stim2 (Stim1/2K14Cre(+)) in salivary glands exhibited reduced Ca2+ levels and hyposalivate. SOCE was functionally required for the activation of the Ca2+ activated Cl- channel ANO1. Ageing Stim1/2K14Cre(+) mice showed no evidence of lymphocytic infiltration or increased levels of autoantibodies characteristic of SjD, possibly associated with a downregulation of toll-like receptor 8 (Tlr8) expression. Salivary gland biopsies of SjD patients showed increased expression of STIM1 and TLR7/8. Our study shows that SOCE activates ANO1 function and fluid secretion in salivary glands and highlights a potential link between SOCE and TLR signaling in SjD.