Absence of Survival and Motor Deficits in 500 Repeat C9ORF72 BAC Mice.

A hexanucleotide repeat expansion at C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD). Initial studies of bacterial artificial chromosome (BAC) transgenic mice harboring this expansion described an absence of motor and survival phenotypes. However, a recent study by Liu and colleagues described transgenic mice harboring a large repeat expansion (C9-500) and reported decreased survival and progressive motor phenotypes. To determine the utility of the C9-500 animals for understanding degenerative mechanisms, we validated and established two independent colonies of transgene carriers. However, extended studies of these animals for up to 1 year revealed no reproducible abnormalities in survival, motor function, or neurodegeneration. Here, we propose several potential explanations for the disparate nature of our findings from those of Liu and colleagues. Resolving the discrepancies we identify will be essential to settle the translational utility of C9-500 mice. This Matters Arising paper is in response to Liu et al. (2016), published in Neuron. See also the response by Nguyen et al. (2020), published in this issue.

C9orf72 suppresses systemic and neural inflammation induced by gut bacteria.

A hexanucleotide-repeat expansion in C9ORF72 is the most common genetic variant that contributes to amyotrophic lateral sclerosis and frontotemporal dementia1,2. The C9ORF72 mutation acts through gain- and loss-of-function mechanisms to induce pathways that are implicated in neural degeneration3-9. The expansion is transcribed into a long repetitive RNA, which negatively sequesters RNA-binding proteins5 before its non-canonical translation into neural-toxic dipeptide proteins3,4. The failure of RNA polymerase to read through the mutation also reduces the abundance of the endogenous C9ORF72 gene product, which functions in endolysosomal pathways and suppresses systemic and neural inflammation6-9. Notably, the effects of the repeat expansion act with incomplete penetrance in families with a high prevalence of amyotrophic lateral sclerosis or frontotemporal dementia, indicating that either genetic or environmental factors modify the risk of disease for each individual. Identifying disease modifiers is of considerable translational interest, as it could suggest strategies to diminish the risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, or to slow progression. Here we report that an environment with reduced abundance of immune-stimulating bacteria10,11 protects C9orf72-mutant mice from premature mortality and significantly ameliorates their underlying systemic inflammation and autoimmunity. Consistent with C9orf72 functioning to prevent microbiota from inducing a pathological inflammatory response, we found that reducing the microbial burden in mutant mice with broad spectrum antibiotics-as well as transplanting gut microflora from a protective environment-attenuated inflammatory phenotypes, even after their onset. Our studies provide further evidence that the microbial composition of our gut has an important role in brain health and can interact in surprising ways with well-known genetic risk factors for disorders of the nervous system.

The C9orf72-interacting protein Smcr8 is a negative regulator of autoimmunity and lysosomal exocytosis.

While a mutation in C9ORF72 is the most common genetic contributor to amyotrophic lateral sclerosis (ALS), much remains to be learned concerning the function of the protein normally encoded at this locus. To elaborate further on functions for C9ORF72, we used quantitative mass spectrometry-based proteomics to identify interacting proteins in motor neurons and found that its long isoform complexes with and stabilizes SMCR8, which further enables interaction with WDR41. To study the organismal and cellular functions for this tripartite complex, we generated Smcr8 loss-of-function mutant mice and found that they developed phenotypes also observed in C9orf72 loss-of-function animals, including autoimmunity. Along with a loss of tolerance for many nervous system autoantigens, we found increased lysosomal exocytosis in Smcr8 mutant macrophages. In addition to elevated surface Lamp1 (lysosome-associated membrane protein 1) expression, we also observed enhanced secretion of lysosomal components-phenotypes that we subsequently observed in C9orf72 loss-of-function macrophages. Overall, our findings demonstrate that C9ORF72 and SMCR8 have interdependent functions in suppressing autoimmunity as well as negatively regulating lysosomal exocytosis-processes of potential importance to ALS.

Monitoring peripheral nerve degeneration in ALS by label-free stimulated Raman scattering imaging.

The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics.

Pharmacological characterisation of anti-inflammatory compounds in acute and chronic mouse models of cigarette smoke-induced inflammation.

BACKGROUND: Candidate compounds being developed to treat chronic obstructive pulmonary disease are typically assessed using either acute or chronic mouse smoking models; however, in both systems compounds have almost always been administered prophylactically. Our aim was to determine whether the prophylactic effects of reference anti-inflammatory compounds in acute mouse smoking models reflected their therapeutic effects in (more clinically relevant) chronic systems. METHODS: To do this, we started by examining the type of inflammatory cell infiltrate which occurred after acute (3 days) or chronic (12 weeks) cigarette smoke exposure (CSE) using female, C57BL/6 mice (n = 7-10). To compare the effects of anti-inflammatory compounds in these models, mice were exposed to either 3 days of CSE concomitant with compound dosing or 14 weeks of CSE with dosing beginning after week 12. Budesonide (1 mg kg-1; i.n., q.d.), roflumilast (3 mg kg-1; p.o., q.d.) and fluvastatin (2 mg kg-1; p.o., b.i.d.) were dosed 1 h before (and 5 h after for fluvastatin) CSE. These dose levels were selected because they have previously been shown to be efficacious in mouse models of lung inflammation. Bronchoalveolar lavage fluid (BALF) leukocyte number was the primary endpoint in both models as this is also a primary endpoint in early clinical studies. RESULTS: To start, we confirmed that the inflammatory phenotypes were different after acute (3 days) versus chronic (12 weeks) CSE. The inflammation in the acute systems was predominantly neutrophilic, while in the more chronic CSE systems BALF neutrophils (PMNs), macrophage and lymphocyte numbers were all increased (p < 0.05). In the acute model, both roflumilast and fluvastatin reduced BALF PMNs (p < 0.01) after 3 days of CSE, while budesonide had no effect on BALF PMNs. In the chronic model, therapeutically administered fluvastatin reduced the numbers of PMNs and macrophages in the BALF (p ≤ 0.05), while budesonide had no effect on PMN or macrophage numbers, but did reduce BALF lymphocytes (p < 0.01). Roflumilast's inhibitory effects on inflammatory cell infiltrate were not statistically significant. CONCLUSIONS: These results demonstrate that the acute, prophylactic systems can be used to identify compounds with therapeutic potential, but may not predict a compound's efficacy in chronic smoke exposure models.

A comparative assessment of gamma-secretase activity in transgenic and non-transgenic rodent brain.

Amyloid-beta (Abeta) deposits are one of the hallmarks of the neuropathological degeneration observed in Alzheimer's disease (AD) and Abeta concentrations have been reported to vary in different brain regions of AD patients. Abeta is produced by the sequential cleavage of amyloid precursor protein (APP) by beta-secretase and gamma-secretase, respectively. Previous studies have shown that over-expression of the gamma-secretase complex leads to increased gamma-secretase proteolytic activity increasing Abeta production. However, it is not known whether brain regions with highest Abeta concentration also express relatively higher levels of gamma-secretase activity. Accordingly, the relationship between Abeta levels and gamma-secretase activity across brain regions was investigated and correlated in the brains of transgenic and non-transgenic rodents commonly used in AD research. The data demonstrated that Abeta levels do vary in different brain regions in both transgenic and non-transgenic mice but are not correlated with regional gamma-secretase activity. Furthermore, this study demonstrated that while mutations in the APP and PS1 sequences affect the absolute Abeta levels this is not reflected in an increase in gamma-secretase proteolytic activity. The data in the current paper indicate that this assay is able to measure the level of gamma-secretase activity in rodent species. Using this methodology will aid our understanding of physiological gamma-secretase function.