Faculty Bibliography

Objectives: This study uses a virtual framework to examine the left maxillary fragment of the juvenile fossil from Mugharet el'Aliya, Morocco, found in association with an Aterian lithic industry. Previously, this fossil had been ascribed to modern humans or the Neanderthal lineage based on its "archaic"/"Neanderthal-like" features and apparent large size. Here, we conducted a novel 3D shape comparative analysis of the maxillary fragment to clarify its taxonomic affinities with regard to its size and ontogeny. Materials and Methods: Eighty Computed Tomography and surface scans representing ontogenetic samples of Homo sapiens and Homo neanderthalensis were used to capture species-specific differences. The toolkit of geometric morphometrics in combination with surface registration and an elastic iterative closest point algorithm were used to create a dataset of meshes with an identical number of corresponding vertices for the maxillae. Multivariate statistics were applied to Procrustes superimposed coordinates derived from the vertices of this dataset. Results: Our analysis showed affinities of the Mugharet el'Aliya individual with our H. sapiens sample, especially with a subadult individual from Qafzeh. No size-independent affinities with Neanderthals of comparable dental age could be identified. Discussion: Our results add to the evidence connecting fossils from western Asia, especially Qafzeh and Skhul, and the North African Aterian. Furthermore, Mugharet el'Aliya adds to our knowledge of the ontogenetic development of adult morphology that is frequently used to characterize hominin groups, for example, Neanderthals and modern humans.

Enamel formation (amelogenesis) is a two-step process whereby crystals partially grow during the secretory stage followed by a significant growth expansion during the maturation stage concurrent with an increase in vectorial Ca²⁺ transport. This requires tight regulation of cytosolic Ca²⁺ (_c Ca²⁺ ) concentration in the enamel forming ameloblasts by controlling Ca²⁺ influx (entry) and Ca²⁺ extrusion (clearance). Gene and protein expression studies suggest that the plasma membrane Ca²⁺ -ATPases (PMCA1-4) are likely involved in _c Ca²⁺ extrusion in ameloblasts, yet no functional analysis of these pumps has been reported nor whether their activity changes across amelogenesis. PMCAs have high Ca²⁺ affinity and low Ca²⁺ clearance which may be a limiting factor in their contribution to enamel formation as maturation stage ameloblasts handle high Ca²⁺ loads. We analyzed PMCA function in rat secretory and maturation ameloblasts by blocking or potentiating these pumps. Low/moderate elevations in _c Ca²⁺ measured using the Ca²⁺ probe Fura-2-AM show that secretory ameloblasts clear Ca²⁺ faster than maturation stage cells through PMCAs. This process was completely inhibited by an external alkaline (pH 9.0) solution or was significantly delayed by the PMCA blockers vanadate and caloxin 1b1. Eliciting higher _c Ca²⁺ transients via the activation of the ORAI1 Ca²⁺ channel showed that the PMCAs of maturation ameloblasts were more efficient. Inhibiting PMCAs decreased the rate of Ca²⁺ influx via ORAI1 but potentiation with forskolin had no effect. Our findings suggest that PMCAs are functional Ca²⁺ pumps during amelogenesis regulating _c Ca²⁺ upon low and/or moderate Ca²⁺ stimulus in secretory stage, thus participating in amelogenesis.

The regulator of calcineurin (RCAN1) has been implicated in the pathogenesis of Down syndrome (DS). Individuals with DS show dental abnormalities for unknown reasons, and RCAN1 levels have been found to be elevated in several tissues of DS patients. A previous microarray analysis comparing cells of the two main formative stages of dental enamel, secretory and maturation, showed a significant increase in RCAN1 expression in the latter. Because the function of RCAN1 during enamel formation is unknown, there is no mechanistic evidence linking RCAN1 with the dental anomalies in individuals with DS. We investigated the role of RCAN1 in enamel by overexpressing RCAN1 in the ameloblast cell line LS8 (LS8^+RCAN1). We first confirmed that RCAN1 is highly expressed in maturation stage ameloblasts by qRT-PCR and used immunofluorescence to show its localization in enamel-forming ameloblasts. We then analyzed cell redox and mitochondrial bioenergetics in LS8^+RCAN1 cells because RCAN1 is known to impact these processes. We show that LS8^+RCAN1 cells have increased reactive oxygen species (ROS) and decreased mitochondrial bioenergetics without changes in the expression of the complexes of the electron transport chain, or in NADH levels. However, LS8^+RCAN1 cells showed elevated mitochondrial Ca²⁺ uptake and decreased expression of several enamel genes essential for enamel formation. These results provide insight into the role of RCAN1 in enamel and suggest that increased RCAN1 levels in the ameloblasts of individuals with DS may impact enamel formation by altering both the redox environment and mitochondrial function, as well as decreasing the expression of enamel-specific genes.

A paper by Sun et al. identified the Ca²⁺-activated Cl^- channel anoctamin 1 or ANO1 (TMEM16A) as an important regulator of osteoclast function by interacting with RANKL activating signaling pathways involved in bone resorption. Although Cl^- transporters (e.g. ClC7, CLIC5) have been known to be involved in the active process of bone resorption, ANO1 appears to control osteoclast differentiation and function to levels beyond those of other Cl^-transporters. Regulating ANO1 function might be a useful target for therapeutics in osteoporosis.

Plasma membrane protein channels provide a passageway for ions to access the intracellular milieu. Rapid entry of calcium ions into cells is controlled mostly by ion channels, while Ca²⁺-ATPases and Ca²⁺ exchangers ensure that cytosolic Ca²⁺ levels ([Ca²⁺]_cyt) are maintained at low (~100 nM) concentrations. Some channels, such as the Ca²⁺-release-activated Ca²⁺ (CRAC) channels and voltage-dependent Ca²⁺ channels (CACNAs), are highly Ca²⁺-selective, while others, including the Transient Receptor Potential Melastatin (TRPM) family, have broader selectivity and are mostly permeable to monovalent and divalent cations. Activation of CRAC channels involves the coupling between ORAI1-3 channels with the endoplasmic reticulum (ER) located Ca²⁺ store sensor, Stromal Interaction Molecules 1-2 (STIM1/2), a pathway also termed store-operated Ca²⁺ entry (SOCE). The TRPM family is formed by 8 members (TRPM1-8) permeable to Mg²⁺, Ca²⁺, Zn²⁺ and Na⁺ cations, and is activated by multiple stimuli. Recent studies indicated that SOCE and TRPM structure-function are interlinked in some instances, although the molecular details of this interaction are only emerging. Here we review the role of TRPM and SOCE in Ca²⁺ handling and highlight the available evidence for this interaction.

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca²⁺ signaling. Quantification of the Ca²⁺ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca²⁺ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca²⁺ (_m Ca²⁺ ) uptake. However, _m Ca²⁺ extrusion via Na⁺ -Li⁺ -Ca²⁺ exchanger was more prominent in maturation. To address if _m Ca²⁺ uptake via the mitochondrial Ca²⁺ uniporter (MCU) played a role in cytosolic Ca²⁺ (_c Ca²⁺ ) buffering, we stimulated Ca²⁺ influx via the store-operated Ca²⁺ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented _c Ca²⁺ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher _m Ca²⁺ uptake in maturation ameloblasts. The uptake of Ca²⁺ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca²⁺ handling in maturation but only upon stimulation of Ca²⁺ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca²⁺ physiology, but also advance the concept that SOCE and _m Ca²⁺ uptake are complementary processes in biological mineralization.

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca²⁺) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Ca_v blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca²⁺]_cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca²⁺ pools, and Ca²⁺ influx by ORAI channels have been associated with the increase in [Ca²⁺]_cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil's effect on [Ca²⁺]_cyt. To address these questions, we monitored changes in [Ca²⁺]_cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca²⁺]_cyt at concentrations above 10 μM (IC₅₀ around 50 μM) and a fast Ca²⁺ increase capacity at 100 μM. We found that inhibiting IP₃ receptors, depleting the ER-Ca²⁺ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca²⁺]_cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca²⁺ influx by store-operated Ca²⁺ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP₃R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

Most cells use calcium (Ca²⁺) as a second messenger to convey signals that affect a multitude of biological processes. The ability of Ca²⁺ to bind to proteins to alter their charge and conformation is essential to achieve its signaling role. Cytosolic Ca²⁺ (_cCa²⁺) concentration is maintained low at ~100 nM so that the impact of elevations in _cCa²⁺ is readily sensed and transduced by cells. However, such elevations in _cCa²⁺ must be transient to prevent detrimental effects. Cells have developed a variety of systems to rapidly clear the excess of _cCa²⁺ including Ca²⁺ pumps, exchangers and sequestering Ca²⁺ within intracellular organelles. This Ca²⁺ signaling toolkit is evolutionarily adapted so that each cell, tissue, and organ can fulfill its biological function optimally. One of the most specialized cells in mammals are the enamel forming cells, the ameloblasts, which also handle large quantities of Ca²⁺. The end goal of ameloblasts is to synthesize, secrete and mineralize a unique proteinaceous matrix without the benefit of remodeling or repair mechanisms. Ca²⁺ uptake into ameloblasts is mainly regulated by the store operated Ca²⁺ entry (SOCE) before it is transported across the polarized ameloblasts to reach the insulated enamel space. Here we review the ameloblasts Ca²⁺ signaling toolkit and address how the common electronegative non-metal fluoride can alter its function, potentially addressing the biology of dental fluorosis.

Recent studies have provided great insight into hominin life history evolution by utilizing incremental lines found in dental tissues to reconstruct and compare the growth records of extant and extinct humans versus other ape taxa. Among the hominins, studies that have examined Retzius periodicity (RP) variation have come to contradictory conclusions in some instances. To clarify RP variation among hominins and better place this variation in its broader evolutionary context, we conduct the most comprehensive analysis of published RP values for hominins and great apes to date. We gathered all available data from the literature on RP data from extant humans, great apes, and fossil hominins and assessed their variation using parametric and nonparametric analyses of variance. We also performed phylogenetic generalized least-squares regressions of RP data for these taxa as well as a larger set of hominoids for which RP data have been published against data for body mass, encephalization, and mean semicircular canal radius (a proxy for metabolic rate). Our results show that modern humans have a mean RP significantly differing from that of other hominins. Pongo also is significantly different from nearly all other taxa in all analyses. Our results also demonstrate that RP variation among hominins scales with respect to body mass, encephalization, and semicircular canal radius similarly to other hominids but that modern humans and Pongo stand out in this regard. Operating within the hypothesis that RP reflects autonomic biorhythms that regulate multiple life history variables, our results reinforce the idea that Homo sapiens has evolved a life history distinct from other hominins, even from other members of Homo, and suggest that many of these life history differences may be driven by hypothalamic output from the brain.