The Role of SOX Transcription Factors in Ageing and Age-Related Diseases.

The quest for eternal youth and immortality is as old as humankind. Ageing is an inevitable physiological process accompanied by many functional declines that are driving factors for age-related diseases. Stem cell exhaustion is one of the major hallmarks of ageing. The SOX transcription factors play well-known roles in self-renewal and differentiation of both embryonic and adult stem cells. As a consequence of ageing, the repertoire of adult stem cells present in various organs steadily declines, and their dysfunction/death could lead to reduced regenerative potential and development of age-related diseases. Thus, restoring the function of aged stem cells, inducing their regenerative potential, and slowing down the ageing process are critical for improving the health span and, consequently, the lifespan of humans. Reprograming factors, including SOX family members, emerge as crucial players in rejuvenation. This review focuses on the roles of SOX transcription factors in stem cell exhaustion and age-related diseases, including neurodegenerative diseases, visual deterioration, chronic obstructive pulmonary disease, osteoporosis, and age-related cancers. A better understanding of the molecular mechanisms of ageing and the roles of SOX transcription factors in this process could open new avenues for developing novel strategies that will delay ageing and prevent age-related diseases.

Current Opportunities for Targeting Dysregulated Neurodevelopmental Signaling Pathways in Glioblastoma.

Glioblastoma (GBM) is the most common and highly lethal type of brain tumor, with poor survival despite advances in understanding its complexity. After current standard therapeutic treatment, including tumor resection, radiotherapy and concomitant chemotherapy with temozolomide, the median overall survival of patients with this type of tumor is less than 15 months. Thus, there is an urgent need for new insights into GBM molecular characteristics and progress in targeted therapy in order to improve clinical outcomes. The literature data revealed that a number of different signaling pathways are dysregulated in GBM. In this review, we intended to summarize and discuss current literature data and therapeutic modalities focused on targeting dysregulated signaling pathways in GBM. A better understanding of opportunities for targeting signaling pathways that influences malignant behavior of GBM cells might open the way for the development of novel GBM-targeted therapies.

Interplay of SOX transcription factors and microRNAs in the brain under physiological and pathological conditions.

Precise tuning of gene expression, accomplished by regulatory networks of transcription factors, epigenetic modifiers, and microRNAs, is crucial for the proper neural development and function of the brain cells. The SOX transcription factors are involved in regulating diverse cellular processes during embryonic and adult neurogenesis, such as maintaining the cell stemness, cell proliferation, cell fate decisions, and terminal differentiation into neurons and glial cells. MicroRNAs represent a class of small non-coding RNAs that play important roles in the regulation of gene expression. Together with other gene regulatory factors, microRNAs regulate different processes during neurogenesis and orchestrate the spatial and temporal expression important for neurodevelopment. The emerging data point to a complex regulatory network between SOX transcription factors and microRNAs that govern distinct cellular activities in the developing and adult brain. Deregulated SOX/microRNA interplay in signaling pathways that influence the homeostasis and plasticity in the brain has been revealed in various brain pathologies, including neurodegenerative disorders, traumatic brain injury, and cancer. Therapeutic strategies that target SOX/microRNA interplay have emerged in recent years as a promising tool to target neural tissue regeneration and enhance neurorestoration. Numerous studies have confirmed complex interactions between microRNAs and SOX-specific mRNAs regulating key features of glioblastoma. Keeping in mind the crucial roles of SOX genes and microRNAs in neural development, we focus this review on SOX/microRNAs interplay in the brain during development and adulthood in physiological and pathological conditions. Special focus was made on their interplay in brain pathologies to summarize current knowledge and highlight potential future development of molecular therapies.

SOX Transcription Factors as Important Regulators of Neuronal and Glial Differentiation During Nervous System Development and Adult Neurogenesis.

The SOX proteins belong to the superfamily of transcription factors (TFs) that display properties of both classical TFs and architectural components of chromatin. Since the cloning of the Sox/SOX genes, remarkable progress has been made in illuminating their roles as key players in the regulation of multiple developmental and physiological processes. SOX TFs govern diverse cellular processes during development, such as maintaining the pluripotency of stem cells, cell proliferation, cell fate decisions/germ layer formation as well as terminal cell differentiation into tissues and organs. However, their roles are not limited to development since SOX proteins influence survival, regeneration, cell death and control homeostasis in adult tissues. This review summarized current knowledge of the roles of SOX proteins in control of central nervous system development. Some SOX TFs suspend neural progenitors in proliferative, stem-like state and prevent their differentiation. SOX proteins function as pioneer factors that occupy silenced target genes and keep them in a poised state for activation at subsequent stages of differentiation. At appropriate stage of development, SOX members that maintain stemness are down-regulated in cells that are competent to differentiate, while other SOX members take over their functions and govern the process of differentiation. Distinct SOX members determine down-stream processes of neuronal and glial differentiation. Thus, sequentially acting SOX TFs orchestrate neural lineage development defining neuronal and glial phenotypes. In line with their crucial roles in the nervous system development, deregulation of specific SOX proteins activities is associated with neurodevelopmental disorders (NDDs). The overview of the current knowledge about the link between SOX gene variants and NDDs is presented. We outline the roles of SOX TFs in adult neurogenesis and brain homeostasis and discuss whether impaired adult neurogenesis, detected in neurodegenerative diseases, could be associated with deregulation of SOX proteins activities. We present the current data regarding the interaction between SOX proteins and signaling pathways and microRNAs that play roles in nervous system development. Finally, future research directions that will improve the knowledge about distinct and various roles of SOX TFs in health and diseases are presented and discussed.

Extract of Herba Anthrisci cerefolii: Chemical Profiling and Insights into Its Anti-Glioblastoma and Antimicrobial Mechanism of Actions.

Anthriscus cerefolium (L.) Hoffm. is a plant traditionally used around the globe since antiquity. Although widely used in many traditional medicines in different cultures, from the scientific point of view it is poorly investigated. Glioblastoma, a tumor type with poor prognosis, is the most common and lethal brain tumor in adults. Current therapeutic strategies for glioblastoma include surgery, radiation and chemotherapy. On the other hand, it has been revealed that patients with cancers are highly susceptible to microbial infections due to the invasive nature of cancer treatment approaches. This study was designed to investigate the chemical profile of herba Anthriscii cerefoli methanolic extract by applying UHPLC-LTQ OrbiTrap MS4 analysis and to analyze its anti-glioblastoma and antimicrobial activities. This study revealed that methanolic extract of herba Anthrisc cerefolii contained phenolic acids and flavonoids, with 32 compounds being identified. Anti-glioblastoma activity was investigated in vitro using A172 glioblastoma cell line. The cytotoxic effects of the extract on A172 cells were compared to the same effect on primary human gingival fibroblast (HGF-1) cells. Decreased rate of proliferation and changes in cell morphology were detected upon treatment of A172 cells with the extract. The antimicrobial activity of extract was tested against Staphylococcus aureus and Candida species. The extract was active against the tested bacterium and yeasts, inhibiting free floating cells and microbial biofilms. This study is the first one to provide a detailed description of the chemical profile of A. cerefolium extract dealing with scientific insights into its anti-glioblastoma and antimicrobial activities.

Subregion-specific Protective Effects of Fluoxetine and Clozapine on Parvalbumin Expression in Medial Prefrontal Cortex of Chronically Isolated Rats.

Dysregulation of GABAergic system is becoming increasingly associated with depression, psychiatric disorder that imposes severe clinical, social and economic burden. Special attention is paid to the fast-spiking parvalbumin-positive (PV+) interneurons, GABAergic neurons which are highly susceptible to redox dysregulation and oxidative stress and implicated in a variety of psychiatric diseases. Here we analyzed the number of PV+ and cleaved caspase-3-positive (CC3+) cells in the rat medial prefrontal cortical (mPFC) subregions following chronic social isolation (CSIS), an animal model of depression and schizophrenia. Also, we examined potential protective effects of antidepressant fluoxetine (FLX) and atypical antipsychotic clozapine (CLZ) on the number of these cells in mPFC subregions, when applied parallel with CSIS in doses that correspond to therapeutically effective ones in patients. Immunofluorescence analysis revealed decreased number of PV+ cells in cingulate cortex area 1, prelimbic area (PrL), infralimbic area (IL) and dorsal peduncular cortex of the mPFC in isolated rats, which coincided with depressive- and anxiety-like behaviors. In addition, CSIS-induced increase in the number of CC3+ cells was detected in aforementioned subregions of mPFC. Treatments with either FLX or CLZ prevented behavioral changes, decrease in PV+ and increase in CC3+ cell numbers in PrL and IL subregions in isolated rats. These results indicate the importance of intact GABAergic signaling in these areas for resistance against CSIS-induced behavioral changes, as well as subregion-specific protective effects of FLX and CLZ in mPFC of CSIS rats.

Early Impairments of Hippocampal Neurogenesis in 5xFAD Mouse Model of Alzheimer's Disease Are Associated with Altered Expression of SOXB Transcription Factors.

Dysregulation of neurogenesis in the subgranular zone (SGZ) of the hippocampal dentate gyrus has been related to cognitive deficits and memory loss in neurodegenerative diseases, such as Alzheimer's disease (AD). Members of the B group of SOX transcription factors play critical roles in regulating neurogenesis in the embryonic and adult nervous system, including maintaining the multipotency, renewal, and cell fate decision of neural stem/progenitor cells. The aim of the present study was to evaluate the expression patterns of selected SOXB proteins in the SGZ, of 8-week-old male and female 5xFAD mice, which represent a transgenic model of AD with a severe and very early development of amyloid pathology. Immunohistochemical analysis showed a significant decrease in the number of cells expressing SOX1, SOX2, and SOX21 transcription factors within the SGZ of 5xFAD mice in comparison to their non-transgenic counterparts which coincidences with reduced number of doublecortin immunoreactive immature neurons found in Tg males. Despite observed changes in expressional pattern of examined SOXB proteins, the proliferative capacity evaluated by the number of Ki-67 immunoreactive cells remained unaffected in transgenic mice of both genders. Based on our results, we suggest that SOXB proteins might be considered as new biomarkers for the detection of early impairments in adult neurogenesis in different animal models or/and new targets in human regenerative medicine.

SOX14 activates the p53 signaling pathway and induces apoptosis in a cervical carcinoma cell line.

SOX14 is a member of the SOX family of transcription factors mainly involved in the regulation of neural development. Recently, it became evident that SOX14 is one of four hypermethylated genes in cervical carcinoma, considered as a tumor suppressor candidate in this type of malignancy. In this paper we elucidated the role of SOX14 in the regulation of malignant properties of cervical carcinoma cells in vitro. Functional analysis performed in HeLa cells revealed that SOX14 overexpression decreased viability and promoted apoptosis through altering the expression of apoptosis related genes. Our results demonstrated that overexpression of SOX14 initiated accumulation of p53, demonstrating potential cross-talk between SOX14 and the p53 signaling pathway. Further analysis unambiguously showed that SOX14 triggered posttranslational modification of p53 protein, as detected by the significantly increased level of phospho-p53 (Ser-15) in SOX14-overexpressing HeLa cells. Moreover, the obtained results revealed that SOX14 activated p53 protein, which was confirmed by elevated p21Waf1/Cip1, a well known target gene of p53. This study advances our understanding about the role of SOX14 and might explain the molecular mechanism by which this transcription factor could exert tumor suppressor properties in cervical carcinoma.

Epigenetic regulation of human SOX3 gene expression during early phases of neural differentiation of NT2/D1 cells.

Sox3/SOX3 is one of the earliest neural markers in vertebrates. Together with the Sox1/SOX1 and Sox2/SOX2 genes it is implicated in the regulation of stem cell identity. In the present study, we performed the first analysis of epigenetic mechanisms (DNA methylation and histone marks) involved in the regulation of the human SOX3 gene expression during RA-induced neural differentiation of NT2/D1 cells. We show that the promoter of the human SOX3 gene is extremely hypomethylated both in undifferentiated NT2/D1 cells and during the early phases of RA-induced neural differentiation. By employing chromatin immunoprecipitation, we analyze several histone modifications across different regions of the SOX3 gene and their dynamics following initiation of differentiation. In the same timeframe we investigate profiles of selected histone marks on the promoters of human SOX1 and SOX2 genes. We demonstrate differences in histone signatures of SOX1, SOX2 and SOX3 genes. Considering the importance of SOXB1 genes in the process of neural differentiation, the present study contributes to a better understanding of epigenetic mechanisms implicated in the regulation of pluripotency maintenance and commitment towards the neural lineage.

SOX18 Is a Novel Target Gene of Hedgehog Signaling in Cervical Carcinoma Cell Lines.

Although there is much evidence showing functional relationship between Hedgehog pathway, in particular Sonic hedgehog, and SOX transcription factors during embryonic development, scarce data are available regarding their crosstalk in cancer cells. SOX18 protein plays an important role in promoting tumor angiogenesis and therefore emerged as a promising potential target in antiangiogenic tumor therapy. Recently it became evident that expression of SOX18 gene in tumors is not restricted to endothelium of accompanying blood and lymphatic vessels, but in tumor cells as well.In this paper we have identified human SOX18 gene as a novel target gene of Hedgehog signaling in cervical carcinoma cell lines. We have presented data showing that expression of SOX18 gene is regulated by GLI1 and GLI2 transcription factors, final effectors of Hedgehog signaling, and that modulation of Hedgehog signaling activity in considerably influence SOX18 expression. We consider important that Hedgehog pathway inhibitors reduced SOX18 expression, thus showing, for the first time, possibility for manipulationwith SOX18 gene expression. In addition, we analyzed the role of SOX18 in malignant potential of cervical carcinoma cell line, and showed that its overexpression has no influence on cells proliferation and viability, but substantially promotes migration and invasion of cells in vitro. Pro-migratory effect of SOX18 suggests its role in promoting malignant spreading, possibly in response to Hedgehog activation.

Crosstalk between SOXB1 proteins and WNT/ß-catenin signaling in NT2/D1 cells.

During early vertebrate embryogenesis, the expression of SOXB1 proteins is precisely regulated by a number of different mechanisms, including Wnt/ß-catenin signaling. This is essential for controlling the balance between stemness and differentiation in embryonic stem cells. In the present study, we analyzed the molecular mechanism of LiCl action in NT2/D1 cells and examined the crosstalk between SOXB1 proteins and Wnt signaling in this model system. We have shown that LiCl increases ß-catenin level, induces its translocation to the nucleus and consequently up-regulates ß-catenin/Tcf-dependent transcription in NT2/D1 cells. Our results also suggest that LiCl treatment leads to increased expression of SOX2 and SOX3 proteins in NT2/D1 cells through activation of canonical Wnt signaling. Finally, we have detected a negative feedback loop between ß-catenin and SOX2 expression in NT2/D1 cells. Since ß-catenin and SOX2 have been linked to processes of self-renewal and pluripotency, our results have implications for future research on the maintenance of stemness and lineage commitment of embryonic stem cells.

The overexpression of SOX2 affects the migration of human teratocarcinoma cell line NT2/D1.

The altered expression of the SOX2 transcription factor is associated with oncogenic or tumor suppressor functions in human cancers. This factor regulates the migration and invasion of different cancer cells. In this study we investigated the effect of constitutive SOX2 overexpression on the migration and adhesion capacity of embryonal teratocarcinoma NT2/D1 cells derived from a metastasis of a human testicular germ cell tumor. We detected that increased SOX2 expression changed the speed, mode and path of cell migration, but not the adhesion ability of NT2/D1 cells. Additionally, we demonstrated that SOX2 overexpression increased the expression of the tumor suppressor protein p53 and the HDM2 oncogene. Our results contribute to the better understanding of the effect of SOX2 on the behavior of tumor cells originating from a human testicular germ cell tumor. Considering that NT2/D1 cells resemble cancer stem cells in many features, our results could contribute to the elucidation of the role of SOX2 in cancer stem cells behavior and the process of metastasis.

The spatiotemporal segregation of GAD forms defines distinct GABA signaling functions in the developing mouse olfactory system and provides novel insights into the origin and migration of GnRH neurons.

Gamma-aminobutyric acid (GABA) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate-limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 coexpressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually nonoverlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia In contrast GAD67 is expressed in a subregion of the nonsensory epithelium/vomeronasal organ epithelium containing the putative Gonadotropin-releasing hormone (GnRH) progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant embryonic GAD (EGAD) concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and postmigratory (GT1-7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock-out embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools.

Quercetin reduces pluripotency, migration and adhesion of human teratocarcinoma cell line NT2/D1 by inhibiting Wnt/ß-catenin signaling.

Quercetin, a bioflavonoid found in plant foods, has a wide range of therapeutic effects. In order to examine the therapeutic potential of quercetin in teratocarcinoma, we used the human teratocarcinoma cell line NT2/D1 as an in vitro model. We have shown that quercetin inhibits the proliferation, adhesion and migration of NT2/D1 cells and downregulates the expression of pluripotency factors SOX2, Oct4 and Nanog. Our results further suggest that the anticancer effect of quercetin against human teratocarcinoma cells is mediated by targeting the canonical Wnt signaling pathway. Quercetin antagonized the Wnt/ß-catenin signaling pathway in NT2/D1 cells by inhibiting ß-catenin nuclear translocation and the consequent downregulation of ß-catenin-dependent transcription. These data suggest that quercetin as a potent inhibitor of Wnt signaling may be an effective therapeutic agent in cancers with aberrant activation of the Wnt pathway.

Expression analysis of SOX14 during retinoic acid induced neural differentiation of embryonal carcinoma cells and assessment of the effect of its ectopic expression on SOXB members in HeLa cells.

SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although the first SOX14 gene in vertebrates was cloned and characterized more than a decade ago and its expression profile during development was revealed in various animal model systems, the role of this gene during neural development is largely unknown. In the present study we analyzed the expression of SOX14 in human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells. We demonstrated that it is expressed in both cell lines and upregulated during retinoic acid induced neural differentiation. We showed that SOX14 was expressed in both neuronal and non-neuronal differentiated derivatives, as revealed by immunocytochemistry. Since it was previously proposed that increased SOXB2 proteins level interfere with the activity of SOXB1 counteracting partners, we compared expression patterns of SOXB members during retinoic acid induction of embryonal carcinoma cells. We revealed that upregulation of SOX14 expression is accompanied by alterations in the expression patterns of SOXB1 members. In order to analyze the potential cross-talk between them, we generated SOX14 expression construct. The ectopic expression of SOX14 was demonstrated at the mRNA level in NT2/D1, P19 and HeLa cells, while an increased level of SOX14 protein was detected in HeLa cells only. By transient transfection experiments in HeLa cells we showed for the first time that ectopic expression of SOX14 repressed SOX1 expression, whereas no significant effect on SOX2, SOX3 and SOX21 was observed. Data presented here provide an insight into SOX14 expression during in vitro neural differentiation of embryonal carcinoma cells and demonstrate the effect of its ectopic expression on protein levels of SOXB members in HeLa cells. Obtained results contribute to better understanding the role of one of the most conserved SOX proteins.

GABAergic signaling in primary lens epithelial and lentoid cells and its involvement in intracellular Ca2+ modulation.

Primary lens epithelial cell (LEC) cultures derived from newborn (P0) and one-month-old (P30) mouse lenses were used to study GABA (gamma-aminobutyric acid) signaling expression and its effect on the intracellular Ca2+ ([Ca2+]i) level. We have found that these cultures express specific cellular markers for lens epithelial and fiber cells, all components of the functional GABA signaling pathway and GABA, thus recapitulating the developmental program of the ocular lens. Activation of both GABA-A and GABA-B receptors (GABAAR and GABABR) with the specific agonists muscimol and baclofen, respectively induces [Ca2+]i transients that could be blocked by the specific antagonists bicuculline and CGP55845 and were dependent on extracellular Ca2+. Bicuculline did not change the GABA-evoked Ca2+ responses in Ca2-containing buffers, but suppressed them significantly in Ca2+-free buffers suggesting the two receptors couple to convergent Ca2+ mobilization mechanisms with different extracellular Ca2+ sensitivity. Prolonged activation of GABABR induced wave propagation of the Ca2+ signal and persistent oscillations. The number of cells reacting to GABA or GABA+bicuculline in P30 mouse LEC cultures expressing predominantly the synaptic type GABAAR did not differ significantly from the number of reacting cells in P0 mouse LEC cultures. The GABA-induced Ca2+ transients in P30 (but not P0) mouse LEC could be entirely suppressed by co-application of bicuculline and CGP55845. The GABA-mediated Ca2+ signaling may be involved in a variety of Ca2+-dependent cellular processes during lens growth and epithelial cell differentiation.

GABA(A) and GABA(B) receptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca(2+).

Gamma-amminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system of vertebrates, serves as an autocrine/paracrine signaling molecule during development, modulating a number of calcium (Ca(2+))-dependent processes, including proliferation, migration, and differentiation, acting via 2 types of GABA receptors (GABARs): ionotropic GABA(A)Rs and metabotropic GABA(B)Rs. Here, we demonstrate that mouse embryonic stem cells (mESCs), which possess the capacity for virtually unlimited self-renewal and pluripotency, synthesize GABA and express functional GABA(A)Rs and GABA(B)Rs, as well as voltage-gated calcium channels (VGCCs), ryanodine receptors (RyRs), and inwardly rectifying potassium (GIRK) channels. On activation, both GABAR types triggered synergistically intracellular calcium rise. Muscimol (a GABA(A)R agonist) induced single Ca(2+) transients involving both VGCC-mediated Ca(2+) influx and intracellular stores, while baclofen (a GABA(B)R agonist) evoked Ca(2+) transients followed by intercellular Ca(2+) waves and oscillations that were resistant to antagonists and entirely dependent on Ca(2+) release from intracellular stores. Prolonged treatment with muscimol slightly inhibited, while baclofen or SR95531 (a GABA(A)R antagonist) significantly facilitated, mESC proliferation. GABA(A)R-specific ligands also induced morphological and gene expression changes indicating a differentiation shift. Our data suggest that the interplay between GABARs and downstream (coupled) effectors differentially modulates mESC proliferation/differentiation through selective activation of second messenger signaling cascades.-Schwirtlich, M., Emri, Z., Antal, K., Máté, Z., Katarova, Z., Szabó, G. GABA(A) and GABA(B) receptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca(2+).

GABA neurotransmitter signaling in the developing mouse lens: dynamic regulation of components and functionality.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the vertebrate nervous system, serves as a signaling molecule modulating diverse processes during embryonic development. Earlier we have demonstrated that different forms of glutamic acid decarboxylase (GAD) are differentially regulated during mouse lens development. Here we show that the developing lens expresses also components of GABA signaling downstream of GAD. Multiple GABA(A) and GABA(B) receptor subunits as well as the GABA transporters show expression profiles highly correlated with the expression of different GADs. GABA receptors (GABAR) and the vesicular GABA transporter localize at the apical/basal membranes of the lens epithelia and differentiating fibers and may be involved in conventional GABAR-mediated signaling, while the membrane GABA transporters may also function as Na(+)/Cl(-)/GABA carriers. The functionality of GABAR was verified by calcium imaging in whole lenses. Our data suggest that GABA synthesized locally by GAD, acts through GABA receptors by modulating the intracellular calcium levels.

GAD isoforms exhibit distinct spatiotemporal expression patterns in the developing mouse lens: correlation with Dlx2 and Dlx5.

Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter of the adult nervous system and its biosynthetic enzyme glutamic acid decarboxylase (GAD) are abundantly expressed in the embryonic nervous system and are involved in the modulation of cell proliferation, migration, and differentiation. Here we describe for the first time the expression of GABA and embryonic and adult GAD isoforms in the developing mouse lens. We show that the GAD isoforms are sequentially induced with specific spatiotemporal profiles: GAD65 and embryonic GAD isoforms prevail in primary fibers, while GAD67 is the predominant GAD expressed in the postnatal secondary fibers. This pattern correlates well with the expression of Dlx2 and Dlx5, known as upstream regulators of GAD. GABA and GAD are most abundant at the tips of elongating fibers and are absent from organelle-free cells, suggesting their involvement is primarily in shaping of the cytoskeleton during fiber elongation stages.