Identification of DLK1-positive cell clusters in the human adrenal cortex and their potential involvement in adrenocortical carcinoma.

PhD thesisThe adrenal glands are vital endocrine organs responsible for the synthesis and secretion of multiple steroids and hormones. They are composed of an inner adrenal medulla and an outer adrenal cortex. The human adrenal cortex is further subdivided into three distinct zones that differ both morphologically and functionally: the Zona Glomerulosa (ZG), the Zona Fasciculata (ZF), and the Zona Reticularis (ZR). In rats, another zone has been identified between the ZG and the ZF, termed the undifferentiated zone (ZU). This zone has been shown to consist of adrenocortical progenitor cells, expressing Sonic Hedgehog and Delta like homologue 1 (Dlk1). The presence and function of the ZU in human adrenals is not known. In this project I studied the expression of stem/progenitor and steroidogenic markers in the human adrenal cortex and identified a novel cell population in the subcapsular region, which is hypothesised to be similar to the ZU in rats. This cell population expressed the atypical Notch ligand Delta-like homologue 1 (DLK1) but not steroidogenic markers (similar to the rat model), and we termed this as DLK1-cell clusters (DCCs). Following assessment of DLK1 expression across normal adrenals from foetuses to ageing adults, DCCs appear to be of layered continuous appearance in foetuses and in younger individuals and become clustered later in life. However, these were found to be different entities to aldosterone producing cell clusters (APCCs), which are precursors of aldosterone producing adenomas (APAs). Since DLK1 has shown involvement in carcinogenesis, I assessed whether it is involved in DLK1 in adrenocortical carcinomas (ACCs). DLK1 was significantly upregulated in all ACC samples analysed compared to normal adrenals. Further in vitro experiments using human adrenocortical cell line H295R, showed that DLK1-expressing cells possess cancer stem cell characteristics. Collectively, these results indicate that DLK1 could be a novel marker of cancer stem cells in adrenocortical carcinoma, which could potentially be used as a biomarker for identification and treatment.Medical Research CouncilQueen Mary University School of Medicine and DentistryRosetrees Trus

LGR4 deficiency results in delayed puberty through impaired Wnt/beta-catenin signaling

The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands, Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/beta-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/beta-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/beta-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.Peer reviewe

Sphingosine-1-phosphate lyase mutations cause primary adrenal insufficiency and steroid-resistant nephrotic syndrome

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1-/- mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1-/- mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism

Peptide Nanofiber Complexes with siRNA for Deep Brain Gene Silencing by Stereotactic Neurosurgery

Peptide nanofibers (PNFs) are one-dimensional assemblies of amphiphilic peptides in a cylindrical geometry. We postulated that peptide nanofibers (PNFs) can provide the tools for genetic intervention and be used for delivery of siRNA, as they can be engineered with positively charged amino acids that can electrostatically bind siRNA. The aim of this work was to investigate the use of PNFs as vectors for siRNA delivery providing effective gene knockdown. We designed a surfactant-like peptide (palmitoyl-GGGAAAKRK) able to self-assemble into PNFs and demonstrated that complexes of PNF:siRNA are uptaken intracellularly and increase the residence time of siRNA in the brain after intracranial administration. The biological activity of the complexes was investigated <i>in vitro</i> by analyzing the down-regulation of the expression of a targeted protein (BCL2), as well as induction of apoptosis, as well as <i>in vivo</i> by analyzing the relative gene expression upon stereotactic administration into a deep rat brain structure (the subthalamic nucleus). Gene expression levels of <i>BCL2</i> mRNA showed that PNF:siBCL2 constructs were able to silence the target <i>BCL2</i> in specific loci of the brain. Silencing of the <i>BCL2</i> gene resulted in ablation of neuronal cell populations, indicating that genetic interventions by PNF:siRNA complexes may lead to novel treatment strategies of CNS pathologies

Sphingosine-1-phosphate lyase mutations cause primary adrenal insufficiency and steroid-resistant nephrotic syndrome

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G\u3eA (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G\u3eA (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1–/– mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1–/– mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism

Pituitary Carcinoma in a Patient with an SDHB Mutation.

We present the first case of pituitary carcinoma occurring in a patient with a succinate dehydrogenase subunit B (SDHB) mutation and history of paraganglioma. She was initially treated for a glomus tumour with external beam radiotherapy. Twenty-five years later, she was diagnosed with a non-functioning pituitary adenoma, having developed bitemporal hemianopia. Recurrence of the pituitary lesion (Ki-67 10% and p53 overexpressed) occurred 5 years after her transsphenoidal surgery, for which she underwent two further operations followed by radiotherapy. Histology showed large cells with vacuolated clear cytoplasm with positive immunostaining for steroidogenic factor 1 (SF1) and negative staining for pituitary hormones. Four years after the pituitary radiotherapy, two metastatic deposits were identified: a foramen magnum lesion and an intradural extra-medullary cervical lesion at the level of C3/C4. There was also significant growth of the primary pituitary lesion with associated visual deterioration. A biopsy of the foramen magnum lesion, demonstrating cells with vacuolated, clear cytoplasm and positive SF1 staining confirmed a pituitary carcinoma, for which she was commenced on temozolomide chemotherapy. There was dramatic clinical improvement after three cycles and reduction in the size of the lesions was observed following six cycles of temozolomide, and further shrinkage after 10 cycles. The plan is for a total of 12 cycles of temozolomide chemotherapy. SDH mutation-related pituitary tumours have an aggressive phenotype which, in this case, led to metastatic disease. SF1 immunostaining was helpful to identify the tissue origin of the metastatic deposit and to confirm the pituitary carcinoma.The Medical College of Saint Bartholomew’s Hospital TrustNIHR (National Institute of Health Research, UK)MRC project grantBritish Neuropathological Societ