Modelling C9orf72 dipeptide repeat proteins of a physiologically relevant size.

C9orf72 expansions are the most common genetic cause of FTLD and MND identified to date. Although being intronic, the expansion is translated into five different dipeptide repeat proteins (DPRs) that accumulate within patients' neurons. Attempts have been made to model DPRs in cell and animals. However, the majority of these use DPRs repeat numbers much shorter than those observed in patients. To address this we have generated a selection of DPR expression constructs with repeat numbers in excess of 1000 repeats, matching what is seen in patients. Small and larger DPRs produce inclusions with similar morphology but different cellular effects. We demonstrate a length dependent effect using electrophysiology with a phenotype only occurring with the longest DPRs. These data highlight the importance of using physiologically relevant repeat numbers when modelling DPRs.

Identification of biological pathways regulated by PGRN and GRN peptide treatments using transcriptome analysis.

Mutations in progranulin (PGRN) have been linked to two neurodegenerative disorders, heterozygote mutations with frontotemporal lobar degeneration (FTLD) and homozygote mutations with neuronal ceroid lipofuscinosis (NCL). Human PGRN is 593aa secreted growth factor, made up of seven and a half repeats of a highly conserved granulin motif that is cleaved to produce the granulin peptides A-G and paragranulin. While it is thought that PGRN protects against neurodegeneration through its role in inflammation and tissue repair, the role of PGRN and granulins in the nervous system is currently unclear. To better understand this, we prepared recombinant PGRN, granulin A-F and paragranulin, and used these to treat differentiated neuronal SH-SY5Y cells. Using RNA sequencing and bioinformatics techniques we investigated the functional effects of PGRN and the individual granulins upon the transcriptome. For PGRN treatment we show that the main effect of short-duration treatments is the down-regulation of transcripts, supporting that signalling pathway induction appears to be dominant effect. Gene ontology analysis, however, also supports the regulation of biological processes such as the spliceosome and proteasome in response to PGRN treatment, as well as the lysosomal pathway constituents such as CHMP1A, further supporting the role of PGRN in lysosomal function. We also show that the response to granulin treatments involves the regulation of numerous non-coding RNA's, and the granulins cluster into groups of similar activity on the basis of expression profile with paragranulin and PGRN having similar expression profiles, while granulins B, D, E and G appear more similar.

Small deletion in C9orf72 hides a proportion of expansion carriers in FTLD.

Frontotemporal lobar degeneration is a highly familial disease and the most common known genetic cause is the repeat expansion mutation in the gene C9orf72. We have identified 2 brothers with an expansion mutation in C9orf72 using Southern blotting that is undetectable using repeat-primed polymerase chain reaction. Sequencing using high concentrations of DNA denaturants of a bacterial artificial chromosome clone obtained from one of the brothers identified a 10-base pair deletion adjacent to the expansion that presumably confers strong secondary structure that interferes with the genotyping. Using an alternative method, we have identified missed expansion carriers in our cohort, and this number has increased by approximately 25%. This observation has important implications for patients undergoing genetic testing for C9orf72.

Pathogenesis/genetics of frontotemporal dementia and how it relates to ALS.

One of the most interesting findings in the field of neurodegeneration in recent years is tfche discovery of a genetic mutation in the C9orf72 gene, the most common mutation found to be causative of sporadic and familial frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS) and concomitant FTD-ALS (DeJesus-Hernandez et al., 2011b; Renton et al., 2011). While clinical and molecular data, such as the identification of TDP-43 being a common pathological protein (Neumann et al., 2006) have hinted at such a link for years, the identification of what was formally known as "the chromosome 9 FTLD-ALS gene" has provided a foundation for better understanding of the relationship between the two. Indeed, it is now recognized that ALS and FTLD-TDP represent a disease spectrum. In this review, we will discuss the current genetic and pathological features of the FTLD-ALS spectrum.

Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72.

BACKGROUND: Cases of Frontotemporal Lobar Degeneration (FTLD) and Motor Neurone Disease (MND) associated with expansions in C9ORF72 gene are characterised pathologically by the presence of TDP-43 negative, but p62 positive, inclusions in granule cells of the cerebellum and in cells of dentate gyrus and area CA4 of the hippocampus. RESULTS: We screened 84 cases of pathologically confirmed FTLD and 23 cases of MND for the presence of p62 positive inclusions in these three brain regions, and identified 13 positive cases of FTLD and 3 of MND. All cases demonstrated expansions in C9ORF72 by Southern blotting where frozen tissues were available. The p62 positive inclusions in both cerebellum and hippocampus were immunostained by antibodies to dipeptide repeat proteins (DPR), poly Gly-Ala (poly-GA), poly Gly-Pro (poly-GP) and poly Gly-Arg (poly-GR), these arising from a putative non-ATG initiated (RAN) sense translation of the GGGGCC expansion. There was also some slight, but variable, immunostaining with poly-AP antibody implying some antisense translation might also occur, though the relative paucity of immunostaining could reflect poor antigen avidity on the part of the antisense antibodies. Of the FTLD cases with DPR, 6 showed TDP-43 type A and 6 had TDP-43 type B histology; one had FTLD-tau with the pathology of corticobasal degeneration. There were no qualitative or quantitative differences in the pattern of immunostaining with antibodies to DPR, or p62, proteins between TDP-43 type A and type B cases. Ratings for frequency of inclusions immunostained by these poly-GA, poly-GP and poly-GR antibodies broadly correlated with those for immunolabelled by p62 antibody in all three regions. CONCLUSION: We conclude that DPR are a major component of p62 positive inclusions in FTLD and MND.