Neural stem cell homeostasis is affected in cortical organoids carrying a mutation in Angiogenin.

Mutations in Angiogenin (ANG) and TARDBP encoding the 43 kDa transactive response DNA binding protein (TDP-43) are associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). ANG is neuroprotective and plays a role in stem cell dynamics in the haematopoietic system. We obtained skin fibroblasts from members of an ALS-FTD family, one with mutation in ANG, one with mutation in both TARDBP and ANG, and one with neither mutation. We reprogrammed these fibroblasts to induced pluripotent stem cells (iPSCs) and generated cortical organoids as well as induced stage-wise differentiation of the iPSCs to neurons. Using these two approaches we investigated the effects of FTD-associated mutations in ANG and TARDBP on neural precursor cells, neural differentiation, and response to stress. We observed striking neurodevelopmental defects such as abnormal and persistent rosettes in the organoids accompanied by increased self-renewal of neural precursor cells. There was also a propensity for differentiation to later-born neurons. In addition, cortical neurons showed increased susceptibility to stress, which is exacerbated in neurons carrying mutations in both ANG and TARDBP. The cortical organoids and neurons generated from patient-derived iPSCs carrying ANG and TARDBP gene variants recapitulate dysfunctions characteristic of frontotemporal lobar degeneration observed in FTD patients. These dysfunctions were ameliorated upon treatment with wild type ANG. In addition to its well-established role during the stress response of mature neurons, ANG also appears to play a role in neural progenitor dynamics. This has implications for neurogenesis and may indicate that subtle developmental defects play a role in disease susceptibility or onset. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The secretion of the angiogenic and neurotrophic factor angiogenin is COPII and microtubule dependent.

The RNaseA superfamily member Angiogenin (ANG) is a secreted protein involved in neovascularization, cell proliferation and stress response. Dysregulation of ANG expression is found in many cancers with poor prognosis and mutations in ANG are associated with neurodegenerative diseases. While the uptake and nuclear translocation of ANG is relatively well characterised, little is known about how it reaches the plasma membrane and its mode of secretion. We generated SH-SY5Y neuroblastoma cell lines constitutively expressing wild type (WT) Hemagglutinin (HA) epitope tagged mouse Ang1 (mAng1), and two amyotrophic lateral sclerosis associated ANG variants (C39W and K40I). Herein, we show that these cell lines secrete mAng1 into the culture media. Using small molecule inhibitors we probed the route taken between the endoplasmic reticulum and trans-Golgi network during secretion and have characterised it as COPII and microtubule dependent. In addition, we show that disruption by the PI3-kinase inhibitor wortmannin of the later stages of transit to the plasma membrane leads to mAng1 trafficking to lysosomal compartments. This suggests an autophagy dependent regulation of secretion.

The catalytic activity and secretion of zebrafish RNases are essential for their in vivo function in motor neurons and vasculature.

Angiogenin (hANG), a member of the Ribonuclease A superfamily has angiogenic, neurotrophic and neuroprotective activities. Mutations in hANG have been found in patients with Amyotrophic lateral sclerosis (ALS). The zebrafish (Danio rerio) rnasel-1, 2 and 3 are orthologues of hANG and of these only Rnasel-1 and Rnasel-2 have been shown to be angiogenic. Herein we show that NCI-65828, a potent and specific small molecule inhibitor of hANG inhibits Rnasel-1 to a similar extent. Treatment of early zebrafish embryos with NCI-65828, or with terrein, a fungal metabolite which prevents the secretion of hANG, resulted in spinal neuron aberrations as well defects in trunk vasculature. Our detailed expression analysis and inhibitor studies suggest that Rnasel-1 plays important roles in neuronal migration and pathfinding as well as in angiogenesis in zebrafish. Our studies suggest the usefulness of the zebrafish as a model to dissect the molecular consequences of the ANG ALS variants.

The cellular uptake of angiogenin, an angiogenic and neurotrophic factor is through multiple pathways and largely dynamin independent.

Angiogenin (ANG), a member of the RNase superfamily (also known as RNase 5) has neurotrophic, neuroprotective and angiogenic activities. Recently it has also been shown to be important in stem cell homeostasis. Mutations in ANG are associated with neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Fronto-temporal dementia (FTD). ANG is a secreted protein which is taken up by cells and translocated to the nucleus. However, the import pathway/s through which ANG is taken up is/are still largely unclear. We have characterised the uptake of ANG in neuronal, astrocytic and microglial cell lines as well as primary neurons and astrocytes using pharmacological agents as well as dominant negative dynamin and Rab5 to perturb uptake and intracellular trafficking. We find that uptake of ANG is largely clathrin/dynamin independent and microtubule depolymerisation has a marginal effect. Perturbation of membrane ruffling and macropinocytosis significantly inhibited ANG uptake suggesting an uptake mechanism similar to RNase A. Our findings shed light on why mutations which do not overtly affect RNase activity but cause impaired localization are associated with neurodegenerative disease.

Embryoid body arrays: Parallel cryosectioning of spheroid/embryoid body samples for medium through-put analysis.

Three dimensional (3D) culture of mammalian cells is emerging as a powerful new tool to understand organogenesis as well as serve as models for diseases with implications for therapeutic evaluation. 3D cultures are referred to variously as spheroid, organoids or embryoid bodies. While many methods exist for large scale production of embryoid bodies or other spheroid cell aggregates, either at controlled sizes using microwell/micropatterned plates or uncontrolled sizes in suspension dishes, very few protocols exist for medium throughput analysis of differentiation at the histological level. We have developed a method which allows for parallel processing, sectioning and analysis of multiple 3D samples (e.g. fixed at different time points, treated with different drugs/growth factors, generated from different cell lines etc.) by double-embedding blocks in a larger array format. Our protocol has few barriers for use and requires only materials commonly found in any lab currently using embedding materials for cryosectioning. Sectioning in parallel allows histological techniques (such as histochemistry, immunostaining or in situ hybridisation) to be performed simultaneously on many samples on a single slide. This reduces slide to slide variation as well as requiring less reagents, fewer consumables with lower time and labour requirements when compared to individually embedded samples.

Dynamic expression of the mouse orthologue of the human amyotropic lateral sclerosis associated gene C9orf72 during central nervous system development and neuronal differentiation.

The hexanucleotide repeat in the first intron of the C9orf72 gene is the most significant cause of amyotropic lateral sclerosis as well as some forms of fronto-temporal dementia. The C9orf72 protein has been previously reported to be expressed in post-mortem human brain as well as in late embryonic and some postnatal stages in mice. Herein, we present a detailed study of the distribution of C9orf72 protein in the embryonic, postnatal and adult mouse brain, spinal cord as well as during the differentiation of P19 embryonal carcinoma cells to neurons including motor neurons. We show that the expression levels of the C9orf72 transcripts in the developing and adult mouse brain as well as in differentiating neurons, are dynamic. Besides the strong expression in the cerebellum and motor cortex reported previously, we show for the first time that C9orf72 is expressed strongly in the olfactory bulb and also in the hippocampus. Our immunostaining data also reveal a hitherto unreported switch in the cellular distribution of C9orf72 from a predominantly cytoplasmic to a nucleo-cytoplasmic distribution during corticogenesis. This switch in distribution was also observed during differentiation of the pluripotent embryonal carcinoma P19 cell line to mature neurons. Our findings have implications for interpreting the pathophysiology caused by the repeat expansions in C9orf72 in mouse models.

PA6 Stromal Cell Co-Culture Enhances SH-SY5Y and VSC4.1 Neuroblastoma Differentiation to Mature Phenotypes.

Neuroblastoma cell lines such as SH-SY5Y have been used for modelling neurodegenerative diseases and for studying basic mechanisms in neuroscience. Since neuroblastoma cells proliferate and generally do not express markers of mature or functional neurons, we exploited a co-culture system with the stromal cell line PA6 to better induce differentiation to a more physiologically relevant status. We found that co-culture of the neuroblastoma cell lines in the presence of neural inducers such retinoic acid was able to generate a high proportion of quiescent neurons with very long neurites expressing differentiation markers. The co-culture system additionally cuts short the time taken to produce a more mature phenotype. We also show the application of this system to study proteins implicated in motor neuron disease.

High-grade Angiosarcoma Associated with Ruptured Breast Implants.

SUMMARY: Since the serendipitous discovery that implanted polymers cause sarcomas in rats, much research has been conducted to prove or disprove a link between silicone breast implants and/or polymer-based materials and breast cancer. In light of an initial report that 35% of rats implanted with a variety of polymers developed fibrosarcomas, we report a case of primary angiosarcoma found in a patient presenting with bilateral rupture of gel-filled breast implants.

Structural and molecular insights into the mechanism of action of human angiogenin-ALS variants in neurons.

Mutations in angiogenin (ANG), a member of the ribonuclease A superfamily, are associated with amyotrophic lateral sclerosis (ALS; sporadic and familial) and Parkinson's disease. We have previously shown that ANG is expressed in neurons during neuro-ectodermal differentiation, and that it has both neurotrophic and neuroprotective functions. Here we report the atomic resolution structure of native ANG and 11 ANG-ALS variants. We correlate the structural changes to the effects on neuronal survival and the ability to induce stress granules in neuronal cell lines. ANG-ALS variants that affect the structure of the catalytic site and either decrease or increase the RNase activity affect neuronal survival. Neuronal cell lines expressing the ANG-ALS variants also lack the ability to form stress granules. Our structure-function studies on these ANG-ALS variants are the first to provide insights into the cellular and molecular mechanisms underlying their role in ALS.

The molecular characterisation of unusual subcutaneous spindle cell lesion of breast.

BACKGROUND: Spindle cell lesions of the breast represent an interesting diagnostic challenge as they comprise a wide range of tumours that are rare. Differentiating dermatofibrosarcoma protuberans (DFSP) from other dermatofibromas using CD34 immunohistochemistry alone is difficult; therefore, fluorescence in situ hybridisation (FISH) analysis is often employed to identify typical COL1A1-PDGFB fusion or gene rearrangement. Although molecular confirmation of diagnosis is unnecessary in the majority of DFSP cases, the detection of chromosomal rearrangement is valuable in tumours that show unusual clinicopathological features as in this study the authors report a case of DFSP of breast that did not show any typical known molecular features. METHODS AND RESULTS: Morphological and immunohistochemical study was highly suggestive of the diagnosis of DFSP. To further investigate this case, DNA copy number alterations were investigated by the 250 K Affymetrix SNP Mapping array. DNA analysis did not show any of the known translocations reported in DFSP or any known solid tumour category. However, in addition to copy number changes on chromosome 1, amplification of chromosome 7p which contains the epidermal growth factor receptor (EGFR) gene was observed. Results from EGFR FISH showed an increase in EGFR gene to chromosome 7 ratio (3:1) suggesting amplification of the EGFR gene. CONCLUSION: This case of an unusual DFSP demonstrates that genomic interrogation provides additional potential targets such as a therapeutic avenue with anti-EGFR therapies and shows the power of molecular characterisation of unusual tumours for a personalised medicine approach.