Unravelling the clinical spectrum and the role of repeat length in C9ORF72 repeat expansions

Since the discovery of the C9orf72 repeat expansion as the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis, it has increasingly been associated with a wider spectrum of phenotypes, including other types of dementia, movement disorders, psychiatric symptoms and slowly progressive FTD. Prompt recognition of patients with C9orf72-associated diseases is essential in light of upcoming clinical trials. The striking clinical heterogeneity associated with C9orf72 repeat expansions remains largely unexplained. In contrast to other repeat expansion disorders, evidence for an effect of repeat length on phenotype is inconclusive. Patients with C9orf72-associated diseases typically have very long repeat expansions, containing hundreds to thousands of GGGGCC-repeats, but smaller expansions might also have clinical significance. The exact threshold at which repeat expansions lead to neurodegeneration is unknown, and discordant cut-offs between laboratories pose a challenge for genetic counselling. Accurate and large-scale measurement of repeat expansions has been severely hindered by technical difficulties in sizing long expansions and by variable repeat lengths across and within tissues. Novel long-read sequencing approaches have produced promising results and open up avenues to further investigate this enthralling repeat expansion, elucidating whether its length, purity, and methylation pattern might modulate clinical features of C9orf72-related diseases

Prosaposin is a regulator of progranulin levels and oligomerization

Progranulin (GRN) loss-of-function mutations leading to progranulin protein (PGRN) haploinsufficiency are prevalent genetic causes of frontotemporal dementia. Reports also indicated PGRN-mediated neuroprotection in models of Alzheimer’s and Parkinson’s disease; thus, increasing PGRN levels is a promising therapeutic for multiple disorders. To uncover novel PGRN regulators, we linked whole-genome sequence data from 920 individuals with plasma PGRN levels and identified the prosaposin (PSAP) locus as a new locus significantly associated with plasma PGRN levels. Here we show that both PSAP reduction and overexpression lead to significantly elevated extracellular PGRN levels. Intriguingly, PSAP knockdown increases PGRN monomers, whereas PSAP overexpression increases PGRN oligomers, partly through a protein–protein interaction. PSAP-induced changes in PGRN levels and oligomerization replicate in human-derived fibroblasts obtained from a GRN mutation carrier, further supporting PSAP as a potential PGRN-related therapeutic target. Future studies should focus on addressing the relevance and cellular mechanism by which PGRN oligomeric species provide neuroprotection

Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers.

Clinical and neuropathological characteristics associated with G4C2 repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, are highly variable. To gain insight on the molecular basis for the heterogeneity among C9ORF72 mutation carriers, we evaluated associations between features of disease and levels of two abundantly expressed "c9RAN proteins" produced by repeat-associated non-ATG (RAN) translation of the expanded repeat. For these studies, we took a departure from traditional immunohistochemical approaches and instead employed immunoassays to quantitatively measure poly(GP) and poly(GA) levels in cerebellum, frontal cortex, motor cortex, and/or hippocampus from 55 C9ORF72 mutation carriers [12 patients with ALS, 24 with frontotemporal lobar degeneration (FTLD) and 19 with FTLD with motor neuron disease (FTLD-MND)]. We additionally investigated associations between levels of poly(GP) or poly(GA) and cognitive impairment in 15 C9ORF72 ALS patients for whom neuropsychological data were available. Among the neuroanatomical regions investigated, poly(GP) levels were highest in the cerebellum. In this same region, associations between poly(GP) and both neuropathological and clinical features were detected. Specifically, cerebellar poly(GP) levels were significantly lower in patients with ALS compared to patients with FTLD or FTLD-MND. Furthermore, cerebellar poly(GP) associated with cognitive score in our cohort of 15 patients. In the cerebellum, poly(GA) levels similarly trended lower in the ALS subgroup compared to FTLD or FTLD-MND subgroups, but no association between cerebellar poly(GA) and cognitive score was detected. Both cerebellar poly(GP) and poly(GA) associated with C9ORF72 variant 3 mRNA expression, but not variant 1 expression, repeat size, disease onset, or survival after onset. Overall, these data indicate that cerebellar abnormalities, as evidenced by poly(GP) accumulation, associate with neuropathological and clinical phenotypes, in particular cognitive impairment, of C9ORF72 mutation carriers

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell-derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.</p

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell-derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.</p

Characterization of FUS Mutations in Amyotrophic Lateral Sclerosis Using RNA-Seq

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in severe muscle weakness and eventual death by respiratory failure. Although little is known about its pathogenesis, mutations in fused in sarcoma/translated in liposarcoma (FUS) are causative for familial ALS. FUS is a multifunctional protein that is involved in many aspects of RNA processing. To elucidate the role of FUS in ALS, we overexpressed wild-type and two mutant forms of FUS in HEK-293T cells, as well as knocked-down FUS expression. This was followed by RNA-Seq to identify genes which displayed differential expression or altered splicing patterns. Pathway analysis revealed that overexpression of wild-type FUS regulates ribosomal genes, whereas knock-down of FUS additionally affects expression of spliceosome related genes. Furthermore, cells expressing mutant FUS displayed global transcription patterns more similar to cells overexpressing wild-type FUS than to the knock-down condition. This observation suggests that FUS mutants do not contribute to the pathogenesis of ALS through a loss-of-function. Finally, our results demonstrate that the R521G and R522G mutations display differences in their influence on transcription and splicing. Taken together, these results provide additional insights into the function of FUS and how mutations contribute to the development of ALS.ALS Foundation NetherlandsAdessium FoundationSeventh Framework Programme (European Commission) (grant number 259867)Thierry Latran FoundationNational Institutes of Health (U.S.) (NIH/NINDS grant R01NS073873)National Institute of Neurological Disorders and Stroke (U.S.) (NIH/NINDS grant numbers 1R01NS065847

Novel clinical associations with specific C9ORF72 transcripts in patients with repeat expansions in C9ORF72

The loss of chromosome 9 open reading frame 72 (C9ORF72) expression, associated with C9ORF72 repeat expansions, has not been examined systematically. Three C9ORF72 transcript variants have been described thus far; the GGGGCC repeat is located between two non-coding exons (exon 1a and exon 1b) in the promoter region of transcript variant 2 (NM_018325.4) or in the first intron of variant 1 (NM_145005.6) and variant 3 (NM_001256054.2). We studied C9ORF72 expression in expansion carriers (n&nbsp;=&nbsp;56) for whom cerebellum and/or frontal cortex was available. Using quantitative real-time PCR and digital molecular barcoding techniques, we assessed total C9ORF72 transcripts, variant 1, variant 2, variant 3, and intron containing transcripts [upstream of the expansion (intron 1a) and downstream of the expansion (intron 1b)]; the latter were correlated with levels of poly(GP) and poly(GA) proteins aberrantly translated from the expansion as measured by immunoassay (n&nbsp;=&nbsp;50). We detected a decrease in expansion carriers as compared to controls for total C9ORF72 transcripts, variant 1, and variant 2: the strongest association was observed for variant 2 (quantitative real-time PCR cerebellum: median 43&nbsp;%, p&nbsp;=&nbsp;1.26e-06, and frontal cortex: median 58&nbsp;%, p&nbsp;=&nbsp;1.11e-05; digital molecular barcoding cerebellum: median 31&nbsp;%, p&nbsp;=&nbsp;5.23e-10, and frontal cortex: median 53&nbsp;%, p&nbsp;=&nbsp;5.07e-10). Importantly, we revealed that variant 1 levels greater than the 25th percentile conferred a survival advantage [digital molecular barcoding cerebellum: hazard ratio (HR) 0.31, p&nbsp;=&nbsp;0.003, and frontal cortex: HR 0.23, p&nbsp;=&nbsp;0.0001]. When focusing on intron containing transcripts, analysis of the frontal cortex revealed an increase of potentially truncated transcripts in expansion carriers as compared to controls [digital molecular barcoding frontal cortex (intron 1a): median 272&nbsp;%, p&nbsp;=&nbsp;0.003], with the highest levels in patients pathologically diagnosed with frontotemporal lobar degeneration. In the cerebellum, our analysis suggested that transcripts were less likely to be truncated and, excitingly, we discovered that intron containing transcripts were associated with poly(GP) levels [digital molecular barcoding cerebellum (intron 1a): r&nbsp;=&nbsp;0.33, p&nbsp;=&nbsp;0.02, and (intron 1b): r&nbsp;=&nbsp;0.49, p&nbsp;=&nbsp;0.0004] and poly(GA) levels [digital molecular barcoding cerebellum (intron 1a): r&nbsp;=&nbsp;0.34, p&nbsp;=&nbsp;0.02, and (intron 1b): r&nbsp;=&nbsp;0.38, p&nbsp;=&nbsp;0.007]. In summary, we report decreased expression of specific C9ORF72 transcripts and provide support for the presence of truncated transcripts as well as pre-mRNAs that may serve as templates for RAN translation. We further show that higher C9ORF72 levels may have beneficial effects, which warrants caution in the development of new therapeutic approaches

Abnormal Expression Of Homeobox Genes And Transthyretin In C9Orf72 Expansion Carriers

Objective: We performed a genome-wide brain expression study to reveal the underpinnings of diseases linked to a repeat expansion in chromosome 9 open reading frame 72 (C9ORF72). Methods: The genome-wide expression profile was investigated in brain tissue obtained from C9ORF72 expansion carriers (n = 32), patients without this expansion (n = 30), and controls (n = 20). Using quantitative real-time PCR, findings were confirmed in our entire pathologic cohort of expansion carriers (n = 56) as well as nonexpansion carriers (n = 31) and controls (n = 20). Results: Our findings were most profound in the cerebellum, where we identified 40 differentially expressed genes, when comparing expansion carriers to patients without this expansion, including 22 genes that have a homeobox (e.g., HOX genes) and/or are located within the HOX gene cluster (top hit: homeobox A5 [HOXA5]). In addition to the upregulation of multiple homeobox genes that play a vital role in neuronal development, we noticed an upregulation of transthyretin (TTR), an extracellular protein that is thought to be involved in neuroprotection. Pathway analysis aligned with these findings and revealed enrichment for gene ontology processes involved in (anatomic) development (e.g., organ morphogenesis). Additional analyses uncovered that HOXA5 and TTR levels are associated with C9ORF72 variant 2 levels as well as with intron-containing transcript levels, and thus, disease-related changes in those transcripts may have triggered the upregulation of HOXA5 and TTR. Conclusions: In conclusion, our identification of genes involved in developmental processes and neuroprotection sheds light on potential compensatory mechanisms influencing the occurrence, presentation, and/or progression of C9ORF72-related diseases