[Expression changes of RNA m6A regulators in mouse cerebellum affected by hypobaric hypoxia stimulation].

Objective: To investigate the role of RNA m6A methylation in mediating cerebellar dysplasia through analyzing the phenotypes of the mouse cerebella and the expression of several key m6A regulators upon hypobaric hypoxia treatment. Methods: Five-day old C57/BL6 mice were exposed to hypobaric hypoxia for 9 days. The status of mouse cerebellar development was analyzed by comparing the body weights, brain weights and histological features. Immunostaining of cell-type-specific markers was performed to analyze the cerebellar morphology. Real-time PCR, Western blot and immunohistochemical staining were performed to detect the expression of key m6A regulators in the mouse cerebella. Results: Compared with the control, the body weights, brain weights and cerebellar volumes of hypobaric hypoxic mice were significantly reduced (P<0.01). The expression of specific markers in different cells, including NeuN (mature neuron), Calbindin-D28K (Purkinje cell) and GFAP (astrocyte), was decreased in hypobaric hypoxic mouse cerebella (P<0.01), accompanied with disorganized cellular structure. The expression of methyltransferase METTL3 was significantly down-regulated in the cerebella of hypobaric hypoxic mice (P<0.05). Conclusions: Hypobaric hypoxia stimulation causes mouse cerebellar dysplasia, with structural abnormalities in mature granular neurons, Purkinje cells and astrocytes. Expression of METTL3 is decreased in hypobaric hypoxic mice cerebellum compared with that of normobaric normoxic mice, suggesting that its mediated RNA m6A methylation may play an important role in hypobaric hypoxia-induced mouse cerebellar dysplasia.

Protein Exaptation by Endogenous Retroviral Elements Shapes Tumor Cell Senescence and Downstream Immune Signaling.

Cancer cell senescence in lung squamous cell carcinoma (LUSC) is associated with a poor response to chemotherapies and immunotherapies due to promotion of an immunosuppressive tumor microenvironment. This environment is shaped by the senescence-associated secretory pathway, which recruits suppressive immune cell populations. In a recent study, Attig and colleagues identified a transcription factor-activated molecular switch that circumvents cellular senescence through increased expression of the calbindin protein. A human endogenous retrovirus (HERV) sequence upstream of the calbindin gene, CALB1, promotes the transcription of an HERVH-CALB1 transcript through a splice event at the third CALB1 exon in a process known as protein exaptation. The KLF5 transcription factor mediates this transcriptional activity by binding at the HERVH sequence, subsequently initiating the chimeric HERVH-CALB1 transcription. This increased expression of calbindin reduces CXCL8 chemokine production and downstream neutrophil recruitment in LUSC tumor cells. CALB1 exaptation by HERVH is one example by which endogenous retroelements (ERE) regulate immunity in human cancers, highlighting the emerging role of EREs in tumor immunity.

Human endogenous retrovirus onco-exaptation counters cancer cell senescence through calbindin.

Increased levels and diversity of human endogenous retrovirus (HERV) transcription characterize most cancer types and are linked with disease outcomes. However, the underlying processes are incompletely understood. Here, we show that elevated transcription of HERVH proviruses predicted survival of lung squamous cell carcinoma (LUSC) and identified an isoform of CALB1, encoding calbindin, ectopically driven by an upstream HERVH provirus under the control of KLF5, as the mediator of this effect. HERVH-CALB1 expression was initiated in preinvasive lesions and associated with their progression. Calbindin loss in LUSC cell lines impaired in vitro and in vivo growth and triggered senescence, consistent with a protumor effect. However, calbindin also directly controlled the senescence-associated secretory phenotype (SASP), marked by secretion of CXCL8 and other neutrophil chemoattractants. In established carcinomas, CALB1-negative cancer cells became the dominant source of CXCL8, correlating with neutrophil infiltration and worse prognosis. Thus, HERVH-CALB1 expression in LUSC may display antagonistic pleiotropy, whereby the benefits of escaping senescence early during cancer initiation and clonal competition were offset by the prevention of SASP and protumor inflammation at later stages.

Juxtacellular opto-tagging of hippocampal CA1 neurons in freely moving mice.

Neural circuits are made of a vast diversity of neuronal cell types. While immense progress has been made in classifying neurons based on morphological, molecular, and functional properties, understanding how this heterogeneity contributes to brain function during natural behavior has remained largely unresolved. In the present study, we combined the juxtacellular recording and labeling technique with optogenetics in freely moving mice. This allowed us to selectively target molecularly defined cell classes for in vivo single-cell recordings and morphological analysis. We validated this strategy in the CA1 region of the mouse hippocampus by restricting Channelrhodopsin expression to Calbindin-positive neurons. Directly versus indirectly light-activated neurons could be readily distinguished based on the latencies of light-evoked spikes, with juxtacellular labeling and post hoc histological analysis providing 'ground-truth' validation. Using these opto-juxtacellular procedures in freely moving mice, we found that Calbindin-positive CA1 pyramidal cells were weakly spatially modulated and conveyed less spatial information than Calbindin-negative neurons - pointing to pyramidal cell identity as a key determinant for neuronal recruitment into the hippocampal spatial map. Thus, our method complements current in vivo techniques by enabling optogenetic-assisted structure-function analysis of single neurons recorded during natural, unrestrained behavior.

A Promising Combination: PACAP and PARP Inhibitor Have Therapeutic Potential in Models of Diabetic and Hypertensive Retinopathies.

Diabetes and hypertension are complex pathologies with increasing prevalence nowadays. Their interconnected pathways are frequently manifested in retinopathies. Severe retinal consequences and their tight connections as well as their possible treatments are particularly important to retinal research. In the present, work we induced diabetes with streptozotocin in spontaneously hypertensive rats and treated them either with PACAP or olaparib and alternatively with both agents. Morphological and immunohistochemical analyses were carried out to describe cell-specific changes during pathologies and after different treatments. Diabetes and hypertension caused massive structural and cellular changes especially when they were elicited together. Hypertension was crucial in the formation of ONL and OPL damage while diabetes caused significant differences in retinal thickness, OPL thickness and in the cell number of the GCL. In diabetes, double neuroprotective treatment ameliorated changes of calbindin-positive cells, rod bipolar cells and dopaminergic amacrine cells. Double treatment was curative in hypertensive diabetic rat retinas, especially in the case of rod bipolar and parvalbumin-positive cells compared to untreated or single-treated retinas. Our results highlighted the promising therapeutic benefits of olaparib and PACAP in these severe metabolic retinal disorders.

Calbindin regulates Kv4.1 trafficking and excitability in dentate granule cells via CaMKII-dependent phosphorylation.

Calbindin, a major Ca2+ buffer in dentate granule cells (GCs), plays a critical role in shaping Ca2+ signals, yet how it regulates neuronal function remains largely unknown. Here, we found that calbindin knockout (CBKO) mice exhibited dentate GC hyperexcitability and impaired pattern separation, which co-occurred with reduced K+ current due to downregulated surface expression of Kv4.1. Relatedly, manipulation of calbindin expression in HT22 cells led to changes in CaMKII activation and the level of surface localization of Kv4.1 through phosphorylation at serine 555, confirming the mechanism underlying neuronal hyperexcitability in CBKO mice. We also discovered that Ca2+ buffering capacity was significantly reduced in the GCs of Tg2576 mice to the level of CBKO GCs, and this reduction was restored to normal levels by antioxidants, suggesting that calbindin is a target of oxidative stress. Our data suggest that the regulation of CaMKII signaling by Ca2+ buffering is crucial for neuronal excitability regulation.

Lycii radicis cortex inhibits glucocorticoid­induced bone loss by downregulating Runx2 and BMP­2 expression.

Lycii radicis cortex (LRC) has been used to regulate high blood pressure, body temperature, pain and bone disorders in East Asia. Glucocorticoids (GCs), also known as steroids, are potent immunity regulators widely used in the treatment of inflammatory diseases. However, despite their effectiveness, GC usage is strictly controlled due to severe side­effects, such as osteoporosis. However, further research is required as to date, at least to the best of our knowledge, there is no appropriate model to overcome secondary osteoporosis as a side­effect of GC use. Thus, the aim of the present study was to establish an experimental model of osteoporosis induced by GC. Furthermore, the present study aimed to establish the research methodology for medical evaluations of the effectiveness and side­effects of GCs. A secondary osteoporosis animal model was established, and the animals were divided into two groups as follows: The allergic contact dermatitis (ACD)­induced group and the non­ACD­induced group. In the ACD­induced group, a GC topical application group was compared with a GC subcutaneous injection group. The results revealed that the presence of ACD affected the induction of GC­mediated osteoporosis. Therefore, the group exhibiting induced ACD that was treated with a topical application of GC was selected for examining the side­effects of GCs. The effects of LRC on secondary osteoporosis were confirmed in vivo and in vitro. The results indicated that LRC regulated dexamethasone­induced osteoblast apoptotic markers, including caspase­6, caspase­9, X­linked inhibitor of apoptosis, apoptosis inhibitor 1 and apoptosis inhibitor 2, and increased the expression of osteoblast differentiation­related genes, such as Runt­related transcription factor 2 and bone morphogenetic protein 2 in the MC3T3E­1 cell line. LRC also significantly reduced GC­induced osteoporosis and exerted anti­inflammatory effects in vivo. In addition, LRC inhibited the reduction of calbindin­D28k in the kidney. Overall, the results of the present study suggest that the use of LRC alleviates GC­induced secondary osteoporosis.

Generation of mature and functional hair cells by co-expression of Gfi1, Pou4f3, and Atoh1 in the postnatal mouse cochlea.

The mammalian cochlea cannot regenerate functional hair cells (HCs) spontaneously. Atoh1 overexpression as well as other strategies are unable to generate functional HCs. Here, we simultaneously upregulated the expression of Gfi1, Pou4f3, and Atoh1 in postnatal cochlear supporting cells (SCs) in vivo, which efficiently converted SCs into HCs. The newly regenerated HCs expressed HC markers Myo7a, Calbindin, Parvalbumin, and Ctbp2 and were innervated by neurites. Importantly, many new HCs expressed the mature and terminal marker Prestin or vesicular glutamate transporter 3 (vGlut3), depending on the subtypes of the source SCs. Finally, our patch-clamp analysis showed that the new HCs in the medial region acquired a large K+ current, fired spikes transiently, and exhibited signature refinement of ribbon synapse functions, in close resemblance to native wild-type inner HCs. We demonstrated that co-upregulating Gfi1, Pou4f3, and Atoh1 enhances the efficiency of HC generation and promotes the functional maturation of new HCs.

Two septal-entorhinal GABAergic projections differentially control coding properties of spatially tuned neurons in the medial entorhinal cortex.

Septal parvalbumin-expressing (PV+) and calbindin-expressing (CB+) projections inhibit low-threshold and fast-spiking interneurons, respectively, in the medial entorhinal cortex (MEC). We investigate how the two inputs control neuronal activity in the MEC in freely moving mice. Stimulation of PV+ and CB+ terminals causes disinhibition of spatially tuned MEC neurons, but exerts differential effects on temporal coding and burst firing. Thus, recruitment of PV+ projections disrupts theta-rhythmic firing of MEC neurons, while stimulation of CB+ projections increases burst firing of grid cells and enhances phase precession in a cell-type-specific manner. Inactivation of septal PV+ or CB+ neurons differentially affects context, reference, and working memory. Together, our results reveal how specific connectivity of septal GABAergic projections with MEC interneurons translates into differential modulation of MEC neuronal coding.

Ablating Adult Neural Stem Cells Improves Synaptic and Cognitive Functions in Alzheimer Models.

Adult neurogenesis is impaired in the hippocampus of patients with Alzheimer disease (AD) as well as AD models. However, it is far from clear how modulating adult neurogenesis affects AD neuropathology. We confirm that adult hippocampal neurogenesis is impaired in two AD models. Surprisingly, however, cognitive functions are improved in AD models after ablating adult neural stem cells (aNSCs). Ablation of aNSCs does not affect the levels of amyloid ß but restores the normal synaptic transmission in the dentate gyrus (DG) granule cells of AD models. Furthermore, calbindin depletion in the DG of AD mice is ameliorated after aNSC ablation, and knocking down calbindin abolishes the effects of aNSC ablation on synaptic and cognitive functions of AD mice. Together, our data suggest that cognitive functions of AD mice are improved after aNSC ablation, which is associated with the restoration of synaptic transmission in the DG granule cells with calbindin as an important mediator.

Benefits and constrains of covalency: the role of loop length in protein stability and ligand binding.

Protein folding is governed by non-covalent interactions under the benefits and constraints of the covalent linkage of the backbone chain. In the current work we investigate the influence of loop length variation on the free energies of folding and ligand binding in a small globular single-domain protein containing two EF-hand subdomains-calbindin D9k. We introduce a linker extension between the subdomains and vary its length between 1 to 16 glycine residues. We find a close to linear relationship between the linker length and the free energy of folding of the Ca2+-free protein. In contrast, the linker length has only a marginal effect on the Ca2+ affinity and cooperativity. The variant with a single-glycine extension displays slightly increased Ca2+ affinity, suggesting that the slightly extended linker allows optimized packing of the Ca2+-bound state. For the extreme case of disconnected subdomains, Ca2+ binding becomes coupled to folding and assembly. Still, a high affinity between the EF-hands causes the non-covalent pair to retain a relatively high apparent Ca2+ affinity. Our results imply that loop length variation could be an evolutionary option for modulating properties such as protein stability and turnover without compromising the energetics of the specific function of the protein.

Stem cell regionalization during olfactory bulb neurogenesis depends on regulatory interactions between Vax1 and Pax6.

Different subtypes of interneurons, destined for the olfactory bulb, are continuously generated by neural stem cells located in the ventricular and subventricular zones along the lateral forebrain ventricles of mice. Neuronal identity in the olfactory bulb depends on the existence of defined microdomains of pre-determined neural stem cells along the ventricle walls. The molecular mechanisms underlying positional identity of these neural stem cells are poorly understood. Here, we show that the transcription factor Vax1 controls the production of two specific neuronal subtypes. First, it is directly necessary to generate Calbindin expressing interneurons from ventro-lateral progenitors. Second, it represses the generation of dopaminergic neurons by dorsolateral progenitors through inhibition of Pax6 expression. We present data indicating that this repression occurs, at least in part, via activation of microRNA miR-7.

Calbindin-D9k is a Novel Risk Gene for Neurodegenerative Disease.

BACKGROUND/AIMS: Calcium homeostasis plays a crucial role in neuronal development and disease. Calbindin-D9k (CaBP-9k) acts as calcium modulators and sensors in various tissues. However, the neurobiological functions of CaBP-9k are unknown. METHODS: We used CaBP-9k knockout (KO) mice to investigate the roles of these gene in neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We used anatomical and biochemical approaches to characterize functional abnormalities of the brain in the CaBP-9k KO mice. RESULTS: We found that the brains of CaBP-9k KO mice have increased APP/ß-amyloid, Tau, and α-synuclein accumulation and endoplasmic reticulum (ER) stress-induced apoptosis. Neurons deficient for these CaBP-9k had abnormal intracellular calcium levels and responses. ER stress inhibitor TUDCA reduced ER stress-induced apoptosis and restored ER stress- and apoptosis-related proteins expression to wild-type levels in CaBP-9k KO mice. Furthermore, treatment with TUDCA rescued the abnormal memory and motor behaviors exhibited by older CaBP-9k KO mice. CONCLUSION: Our results suggest that a loss of CaBP-9k may contribute to the onset and progression of neurodegenerative diseases.

Depression and Temporal Lobe Epilepsy: Expression Pattern of Calbindin Immunoreactivity in Hippocampal Dentate Gyrus of Patients Who Underwent Epilepsy Surgery with and without Comorbid Depression.

PURPOSE: Changes in calbindin (CB) expression have been reported in patients with temporal lobe epilepsy (TLE) with controversial implications on hippocampal functions. The aim of this study was to determine the CB immunoreactivity in hippocampal dentate gyrus of patients who underwent epilepsy surgery for drug-resistant TLE with and without comorbid depression and/or memory deficits. METHODS: Selected hippocampal samples from patients with TLE who underwent epilepsy surgery were included. Clinical and complementary assessment: EEG, video-EEG, MRI, psychiatric assessment (structured clinical interview, DSM-IV), and memory assessment (Rey auditory verbal learning test, RAVLT; Rey-Osterrieth complex figure test, RCFT), were determined before surgery. Hippocampal sections were processed using immunoperoxidase with the anti-calbindin antibody. The semiquantitative analysis of CB immunoreactivity was determined in dentate gyrus by computerized image analysis (ImageJ). RESULTS: Hippocampal sections of patients with TLE and HS (n = 24) and postmortem controls (n = 5) were included. A significant reduction of CB+ cells was found in patients with TLE (p < 0.05, Student's t-test). Among TLE cases (n = 24), depression (n = 12) and memory deficit (n = 17) were determined. Depression was associated with a higher % of cells with the CB dendritic expression (CB-sprouted cells) (F(1, 20) = 11.81, p = 0.003, hp2 = 0.37), a higher CB+ area (µm2) (F(1, 20) = 5.33, p = 0.032, hp2 = 0.21), and a higher optical density (F(1, 20) = 15.09, p = 0.001, hp2 = 0.43) (two-way ANOVA). The GAF scale (general assessment of functioning) of DSM-IV inversely correlated with the % of CB-sprouted cells (r = -0.52, p = 0.008) and with the CB+ area (r = -0.46, p = 0.022). CONCLUSIONS: In this exploratory study, comorbid depression was associated with a differential pattern of CB cell loss in dentate gyrus combined with a higher CB sprouting. These changes may indicate granular cell dysmaturation associated to the epileptic hyperexcitability phenomena. Further investigations should be carried out to confirm these preliminary findings.

Age-Dependent Calcium-Binding Protein Expression in the Spiral Ganglion and Hearing Performance of C57BL/6J and 129/SvJ Mice.

BACKGROUND/AIMS: Calcium-binding proteins in neurons buffer intracellular free Ca2+ ions, which interact with proteins controlling enzymatic and ion channel activity. The heterogeneous distribution of calretinin, calbindin, and parvalbumin influences calcium homeostasis, and calcium-related neuronal processes play an important role in neuronal aging and degeneration. This study evaluated age-related changes in calretinin, calbindin, and parvalbumin immune reactivity in spiral ganglion cells. METHODS: A total of 16 C57BL/6J and 16 129/SvJ mice at different ages (2, 4, 7, and 12 months) were included in the study. Hearing thresholds were assessed using auditory brainstem response before inner ears were excised for further evaluation. Semiquantitative immunohistochemistry for the aforementioned calcium-binding proteins was performed at the cellular level. RESULTS: The hearing thresholds of C57BL/6J and 129/SvJ mice increased significantly by 7 months of age. The average immune reactivity of calbin-din as well as the relative number of positive cells increased significantly with aging, but no significant alterations in calretinin or parvalbumin were observed. CONCLUSIONS: Upregulation of calbindin could serve as a protection to compensate for functional deficits that occur with aging. Expression of both calretinin and parvalbumin seem to be stabilizing factors in murine inner ears up to the age of 12 months in C57BL/6J and 129/SvJ mice.

The lower expression of parvalbumin in the primary somatosensory cortex of WAG/Rij rats may facilitate the occurrence of absence seizures.

Absence epilepsy (AE) is classified as a genetic generalized epilepsies. WAG/Rij strain of rats are regarded one of the most validated models of absence epilepsy. Studies point out the existence of hyperexcitable focus in somatosensory cortex of these rats, which has been attributed to the deficits in the GABAergic system. In the current study, we studied the changes of calcium binding proteins (CaBPs) in somatosensory cortex (S1) of the 2 and 8 month-old WAG/Rij rats and their age-matched Wistar Albino controls by investigating the expression levels of CaBPs (calbindin, calretinin and parvalbumin) in western blotting. Since WAG/Rij rats showed the low expression level of parvalbumin (PV) in western blots in comparison to Wistar Albino rats, we selectively investigated the number of PV positive neurons using the immunofluorescence staining method in order to confirm this decrement in the perioral region of somatosensory cortex (S1po). The most critical finding of this study was the age- independent reduction in the expression level of PV in the somatosensory cortex of epileptic rats as demonstrating western blotting. Nevertheless, no significant difference was found among numbers of PV + neuron in the S1po region by immunofluorescence staining concerning both of age and strain dependency. These results suggest that the disruption in the activity of the PV-expressing GABAergic interneurons might be involved in the generation of rather than the age-dependent increase in the SWDs in WAG/Rij rats.

Calcium-Binding Proteins as Determinants of Central Nervous System Neuronal Vulnerability to Disease.

Neuronal subpopulations display differential vulnerabilities to disease, but the factors that determine their susceptibility are poorly understood. Toxic increases in intracellular calcium are a key factor in several neurodegenerative processes, with calcium-binding proteins providing an important first line of defense through their ability to buffer incoming calcium, allowing the neuron to quickly achieve homeostasis. Since neurons expressing different calcium-binding proteins have been reported to be differentially susceptible to degeneration, it can be hypothesized that rather than just serving as markers of different neuronal subpopulations, they might actually be a key determinant of survival. In this review, we will summarize some of the evidence that expression of the EF-hand calcium-binding proteins, calbindin, calretinin and parvalbumin, may influence the susceptibility of distinct neuronal subpopulations to disease processes.

Expression of Ca2+-Binding Buffer Proteins in the Human and Mouse Retinal Neurons.

Ca2+-binding buffer proteins (CaBPs) are widely expressed by various neurons throughout the central nervous system (CNS), including the retina. While the expression of CaBPs by photoreceptors, retinal interneurons and the output ganglion cells in the mammalian retina has been extensively studied, a general description is still missing due to the differences between species, developmental expression patterns and study-to-study discrepancies. Furthermore, CaBPs are occasionally located in a compartment-specific manner and two or more CaBPs can be expressed by the same neuron, thereby sharing the labor of Ca2+ buffering in the intracellular milieu. This article reviews this topic by providing a framework on CaBP functional expression by neurons of the mammalian retina with an emphasis on human and mouse retinas and the three most abundant and extensively studied buffer proteins: parvalbumin, calretinin and calbindin.

Vitamin K Supplementation Modulates Bone Metabolism and Ultra-Structure of Ovariectomized Mice.

BACKGROUND/AIMS: Osteoporosis is a bone metabolic disease that affects mostly post-menopausal women. There has been shown that vitamin K (VK) supplementation during menopause may decrease bone loss as well as risk of bone breaking. Aiming to clarify the beneficial role of VK in bone metabolism during menopause, we investigated mineral metabolism and bone ultrastructure of ovariectomized (OVX) mice. METHODS: To determine the effects chronic use of VK in bone structure and mineral metabolism in OVX mice, we used several methods, such as DXA, µCTScan, and SEM as well as biomolecular techniques, such as ELISA and qRT-PCR. In addition, complete analysis of serum hormonal and other molecules associated to bone and lipid metabolism were evaluated overview the effects of VK in menopause murine model. RESULTS: VK treatment significantly affects Pi metabolism independently of OVX, changing Pi plasma, urinary output, balance, and Pi bone mass. Interestingly, VK also increased VLDL in mice independently of castration. In addition, VK increased compact bone mass in OVX mice when we evaluated it by DXA, histomorphometry, µCTScanning. VK increased bone formation markers, osteocalcin, HYP- osteocalcin, and AP whereas it decreased bone resorption markers, such as urinary DPD/creatinine ratio and plasmatic TRAP. Surprisingly, SEM images revealed that VK treatment led to amelioration of microfractures observed in OVX untreated controls. In addition, SHAM operated VK treated mice exhibited higher number of migrating osteoblasts and in situ secretion of AP. OVX led to decreased to in situ secretion of AP that was restored by VK treatment. Moreover, VK treatment increased mRNA expression of bone Calbindin 28KDa independently of OVX. CONCLUSION: VK treatment in OVX mice exhibited beneficial effects on bone ultrastructure, mostly by altering osteoblastic function and secretion of organic bone matrix. Therefore, VK could be useful to treat osteopenic/osteoporotic patients.

Isx9 Regulates Calbindin D28K Expression in Pancreatic ß Cells and Promotes ß Cell Survival and Function.

Pancreatic ß-cell dysfunction and death contribute to the onset of diabetes, and novel strategies of ß-cell function and survival under diabetogenic conditions need to be explored. We previously demonstrated that Isx9, a small molecule based on the isoxazole scaffold, drives neuroendocrine phenotypes by increasing the expression of genes required for ß-cell function and improves glycemia in a model of ß cell regeneration. We further investigated the role of Isx9 in ß-cell survival. We find that Isx9 drives the expression of Calbindin-D28K (D28K), a key regulator of calcium homeostasis, and plays a cytoprotective role through its calcium buffering capacity in ß cells. Isx9 increased the activity of the calcineurin (CN)/cytoplasmic nuclear factor of the activated T-cells (NFAT) transcription factor, a key regulator of D28K, and improved the recruitment of NFATc1, cAMP response element-binding protein (CREB), and p300 to the D28K promoter. We found that nutrient stimulation increased D28K plasma membrane enrichment and modulated calcium channel activity in order to regulate glucose-induced insulin secretion. Isx9-mediated expression of D28K protected ß cells against chronic stress induced by serum withdrawal or chronic inflammation by reducing caspase 3 activity. Consequently, Isx9 improved human islet function after transplantation in NOD-SCID mice in a streptozotocin-induced diabetes model. In summary, Isx9 significantly regulates expression of genes relevant to ß cell survival and function, and may be an attractive therapy to treat diabetes and improve islet function post-transplantation.