Dataset Information

C9ORF72 expression and cellular localization over mouse development.

ABSTRACT: A majority of familial frontotemporal lobar dementia and amyotrophic lateral sclerosis cases are associated with a large repeat expansion in a non-coding region of the C9ORF72 gene. Currently, little is known about the normal function and the expression pattern of the C9ORF72 protein. The aims of this study were to characterize the expression pattern and cellular localization of the three reported mouse isoforms of C9orf72, over a developmental time-course in primary cultured cortical neurons and brain tissue from C57BL/6 mice.We demonstrated that the different isoforms of C9ORF72 at the mRNA and protein level undergo alterations in expression during development and into adulthood. Cellular fractionation and immunofluorescence demonstrated that levels of nuclear and cytoplasmic expression of isoforms changed significantly over the time course. Additionally, immunofluorescence studies showed C9ORF72 labeling as puncta throughout neurons, extending beyond the microtubule cytoskeleton into actin-rich structures such as filopodia and growth cones. Finally, synaptosome preparations demonstrated the presence of C9ORF72 isoform 1 in synaptic-rich fractions from adult mouse brain.In summary, the presence of C9ORF72 as puncta and within synaptic-rich fractions may indicate involvement at the synapse and differential expression of isoforms in nuclei and cytoplasm may suggest distinct roles for the isoforms. Determining the physiological role of C9ORF72 protein may help to determine the role it plays in disease.

SUBMITTER: Atkinson RA

PROVIDER: S-EPMC4582620 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

C9ORF72 expression and cellular localization over mouse development.

Atkinson Rachel A K RA Fernandez-Martos Carmen M CM Atkin Julie D JD Vickers James C JC King Anna E AE

Acta neuropathologica communications 20150925

<h4>Introduction</h4>A majority of familial frontotemporal lobar dementia and amyotrophic lateral sclerosis cases are associated with a large repeat expansion in a non-coding region of the C9ORF72 gene. Currently, little is known about the normal function and the expression pattern of the C9ORF72 protein. The aims of this study were to characterize the expression pattern and cellular localization of the three reported mouse isoforms of C9orf72, over a developmental time-course in primary culture ...[more]

PMID: 26408000

Similar Datasets

Project description:Recent studies have provided evidence that a local renin-angiotensin system (RAS) exists in the retina and plays an important role in retinal neurovascular function. We have recently shown that increased expression of ACE2 and angiotensin (1-7) [Ang (1-7)], two components of the protective axis of the RAS, in the retina via adeno-associated virus (AAV)-mediated gene delivery, conferred protection against diabetes-induced retinopathy. We hypothesized that the protective molecular and cellular mechanisms of Ang (1-7) are mediated by its receptor, Mas, and the expression level and cellular localization dictate the response to Ang (1-7) and activation of subsequent protective signaling pathways. We tested this hypothesis by examining the expression and cellular localization of the Mas receptor in adult and developing mouse retinas.The cellular localization of the Mas receptor protein was determined with immunofluorescence of the eyes of adult and postnatal day 1 (P1), P5, P7, P15, and P21 mice using the Mas receptor-specific antibody, and mRNA was detected with in situ hybridization of paraffin-embedded sections. Western blotting and real-time reverse-transcription (RT)-PCR analysis were performed to determine the relative levels of the Mas protein and mRNA in adult and developing retinas, as well as in cultured retinal Müller glial and RPE cells.In the adult eye, the Mas receptor protein was abundantly present in retinal ganglion cells (RGCs) and photoreceptor cells; a lower level of expression was observed in endothelial cells, Müller glial cells, and other neurons in the inner nuclear layer of the retina. In the developing retina, Mas receptor mRNA and protein expression was detected in the inner retina at P1, and the expression levels increased with age to reach the adult level and pattern by P15. In the adult mouse retina, Mas receptor mRNA was expressed at a much higher level when compared to angiotensin II (Ang II) type I (AT1R) and type II (AT2R) receptor mRNA.The Mas receptor is expressed in developing and adult mouse retinas, and is more abundant in retinal neurons than in endothelial and Müller glial cells. These observations suggest that Mas receptor-mediated signaling may play important roles that extend beyond mediating the vascular effects of Ang (1-7) in developing and adult retinas. In addition, the relatively high expression of the Mas receptor when compared to AT1R suggests that they may play a more important role in maintaining normal retinal physiology than previously considered.

Project description:Hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, but the pathogenic mechanism of this mutation remains unresolved. Haploinsufficiency has been proposed as one potential mechanism. However, insights if and how reduced C9orf72 proteins levels might contribute to disease pathogenesis are still limited because C9orf72 expression, localization and functions in the central nervous system (CNS) are uncertain, in part due to the poor specificity of currently available C9orf72 antibodies.Here, we generated and characterized novel knock-out validated monoclonal rat and mouse antibodies against C9orf72. We found that C9orf72 is a low abundant, cytoplasmic, highly soluble protein with the long 481 amino acid isoform being the predominant, if not exclusively, expressed protein isoform in mouse tissues and human brain. As consequence of the C9orf72 repeat expansion, C9orf72 protein levels in the cerebellum were reduced to 80% in our series of C9orf72 mutation carriers (n?=?17) compared to controls (n?=?26). However, no associations between cerebellar protein levels and clinical phenotypes were seen. Finally, by utilizing complementary immunohistochemical and biochemical approaches including analysis of human iPSC derived motor neurons, we identified C9orf72, in addition to its association to lysosomes, to be localized to the presynapses and able to interact with all members of the RAB3 protein family, suggestive of a role for C9orf72 in regulating synaptic vesicle functions by potentially acting as guanine nucleotide exchange factor for RAB3 proteins.In conclusion, our findings provide further evidence for haploinsufficiency as potential mechanism in C9orf72 pathogenesis by demonstrating reduced protein levels in C9orf72 mutation carriers and important novel insights into the physiological role of C9orf72 in the CNS. Moreover, the described novel monoclonal C9orf72 antibodies will be useful tools to further dissect the cellular and molecular functions of C9orf72.

Project description:A hexanucleotide repeat expansion in a noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reduction of select or total C9orf72 transcript and protein levels is observed in postmortem C9-ALS/FTD tissue, and loss of C9orf72 orthologues in zebrafish and C. elegans results in motor deficits. However, how the reduction in C9orf72 in ALS and FTD might contribute to the disease process remains poorly understood. It has been shown that C9orf72 interacts and forms a complex with SMCR8 and WDR41, acting as a guanine exchange factor for Rab GTPases. Given the known synaptosomal compartmentalization of C9orf72-interacting Rab GTPases, we hypothesized that C9orf72 localization to synaptosomes would be required for the regulation of Rab GTPases and receptor trafficking. This study combined synaptosomal and post-synaptic density preparations together with a knockout-confirmed monoclonal antibody for C9orf72 to assess the localization and role of C9orf72 in the synaptosomes of mouse forebrains. Here, we found C9orf72 to be localized to both the pre- and post-synaptic compartment, as confirmed by both post-synaptic immunoprecipitation and immunofluorescence labelling. In C9orf72 knockout (C9-KO) mice, we demonstrated that pre-synaptic Rab3a, Rab5, and Rab11 protein levels remained stable compared with wild-type littermates (C9-WT). Strikingly, post-synaptic preparations from C9-KO mouse forebrains demonstrated a complete loss of Smcr8 protein levels, together with a significant downregulation of Rab39b and a concomitant upregulation of GluR1 compared with C9-WT mice. We confirmed the localization of Rab39b downregulation and GluR1 upregulation to the dorsal hippocampus of C9-KO mice by immunofluorescence. These results indicate that C9orf72 is essential for the regulation of post-synaptic receptor levels, and implicates loss of C9orf72 in contributing to synaptic dysfunction and related excitotoxicity in ALS and FTD.

Dataset Information

C9ORF72 expression and cellular localization over mouse development.

Publications

C9ORF72 expression and cellular localization over mouse development.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets