Abstract Body

Repeat associated non-AUG (RAN) proteins have been found in a growing number of neurological diseases. We recently showed that targeting RAN proteins using antibodies or decreasing RAN translation with PKR-K296R or metformin improved behavior and increased motor neuron survival in C9orf72 ALS/FTD BAC transgenic mice. These data demonstrate RAN proteins play a central role in C9orf72 ALS/FTD and identify novel strategies to treat RAN-protein diseases.

We now show novel RAN proteins accumulate in spinocerebellar ataxia type 1, 2, 3, 6 and 7 brains. The growing number of RAN-protein diseases and the high percentage (~50%) of repetitive DNA in the human genome raises the possibility that RAN proteins contribute to additional common neurodegenerative diseases. We developed a novel approach that enables identification of microsatellite expansion mutations directly from patient samples. This Cas9-based repeat enrichment and detection (dCas9READ) method, uses RAN-protein aggregate signatures and deactivated clustered regularly interspaced short palindromic repeat associated protein 9 (dCas9) to pull-down candidate RAN-protein producing expansion mutations. dCas9READ works on the principle that expansion mutations provide additional binding sites for repeat-containing single guide RNA (sgRNA)-dCas9 complexes compared to normal alleles. Repeat expansions are enriched using biotin-streptavidin and identified by sequencing. dCas9READ successfully enriched C9orf72 G4C2 (n=4) and DM2 CCTG (n=4) expansion mutations and their corresponding flanking sequences directly from the genomic DNA of all eight patient samples but none of the six expansion-negative controls. These data establish dCas9READ as a novel tool that can be used to identify novel RAN protein diseases that may respond to metformin or other therapies that target RAN translation.

Aims

The aim was to identify alterations in oligodendrocyte cells in frontotemporal dementia caused by C9orf72 expansion (FTLD-C9).

Methods

Using conventional immunohistochemistry we characterized the pathological mechanisms associated with C9orf72 expansion (dipeptide proteins repeat, polyGA, polyGP and polyGR, RNA foci and TDP-43 pathology). We also performed neuropathological studies for Myelin Basic Protein (MBP), a major constituent of the CNS myelin, in the frontal cortex and hippocampus of FTLD-C9orf72 cases to determine a potential specific phenotype for oligodendrocyte cells. We used postmortem samples of patients with a C9orf72 expansion and confirmed FTLD-TDP pathology (n=22), sporadic FTLD-TDP (n=8) and healthy controls without neuropathological alterations (n=10). To quantify the immunoreactivity of MBP we used an in-house semi-automated algorithm. We also performed Western Blot experiments to determine the protein levels of MBP in these three different pathological groups.

Results

We found a significant decrease of MBP immunoreactivity in the grey and white matter of the frontal cortex in the FTLD-C9 group compared to sporadic FTLD-TDP and healthy control groups. We did not find a correlation between MBP expression and the number of DPRs or RNA foci. We observed a trend towards a negative correlation between MBP and TDP-43 pathology in the frontal cortex. We also found a decrease in the MBP protein levels in the grey matter of the frontal cortex in the FTLD-C9 group.

Conclusions

Our results suggest an oligodendroglial dysfunction associated with the C9orf72 expansion. Oligodendroglial dysfunction might constitute an important and so far neglected pathophysiological event.

Aims

A repeat expansion in C9orf72 is the major cause of both frontotemporal dementia (FTD) and motor neuron disease (MND). The expansion is translated to produce 5 dipeptides (DPRs), which aggregate in neurons and are toxic in cell and animal models. However, the mechanisms underlying this toxicity remain unclear.

The NLRP3 inflammasome is a multiprotein complex which forms in immune cells in response to various stimuli, including aggregating proteins such as amyloidß. Inflammasome activation causes release of interleukin-1ß (IL-1ß), triggering an inflammatory response. Interestingly, inflammasome inhibition rescues cognition in rodent models of Alzheimer’s disease, suggesting the inflammasome contributes to disease pathogenesis. Since neuroinflammation is also a feature of FTD, we investigated whether DPRs activate the inflammasome.

Methods

Primary mouse macrophages/microglia were treated with DPRs and inflammasome activation was quantified by IL-1ß ELISA of the media and Western blotting for IL-1ß and caspase-1.

Results

One of the DPRs, GR, caused IL-1ß release and caspase-1 activation in macrophages and microglia from wild-type, but not NLRP3-/- mice, indicating that this was mediated by the inflammasome. Furthermore, drugs which inhibit phagocytosis or cathepsins partially prevented this response. This suggests that GR is phagocytosed into lysosomes which rupture, leading to inflammasome activation. DPR-induced inflammasome activation was blocked by high concentration K⁺ in the media, indicating that K⁺ efflux from the cell is essential for this response.

Conclusions

We demonstrate that C9orf72 dipeptides activate the NLRP3 inflammasome, likely contributing to the neuroinflammation observed in patient brain. These findings highlight the inflammasome as a potential therapeutic target in C9orf72-linked FTD/MND.

Aims

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse, multi-functional family of RNA-binding proteins. Many such proteins, including TDP-43 and FUS, have been implicated in the pathogenesis of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). By contrast hnRNP K has been underexplored in neurodegenerative disease. We sought to perform a comprehensive pathological assessment of hnRNP K protein’s neuronal localisation profile in FTLD, ALS and control brain tissue.

Methods

HnRNP K neuronal localisation was pathologically analysed in the frontal cortex of FTLD-TDP type A (n=28), type C (n=12), FTLD-Tau (n=9), ALS (n=7) and control (n=35) brain. Immunohistochemically stained hnRNP K sections were digitally scanned. Neurons with normally localised (predominantly nuclear) hnRNP K were classified by an automated analytical pipeline utilising deep learning. Abnormally localised neurons (nuclear depletion and cytoplasmic accumulation) were counted manually.

Results

Cytoplasmic mislocalisation of hnRNP K was observed in many cases of FTLD and in a few elderly controls. FTLD-TDP A and FTLD-Tau frontal regions exhibited significantly less normally localised hnRNP K (p=0.01) and significantly more abnormally localised hnRNP K (p<0.01) than controls. Mislocalisation frequency was found to correlate with age at death across the cohort (r=0.44, p<0001). However, the median value of total mislocalisation found in the FTLD-TDP A group was observed 18 years in advance of controls (68 vs 86 years old).

Conclusions

Neuronal mislocalisation of hnRNP K is an entirely novel pathological event to be described in the context of FTLD or ageing. We provide evidence that hnRNP K mislocalisation is related to both processes in support of further investigations.

Aims

Rare genetic causes of human disease have the potential to reveal mechanistic insights into sporadic disease. Identifying novel genetic forms of tauopathy and understanding the pathophysiologic mechanisms that lead to neurofibrillary degeneration may lead to novel insights and perhaps novel therapeutic anti-tau targets.

Methods

Genetic sequencing, clinical phenotyping, neuropathology and radiologic studies were used to define the clinicopathologic features of disease. Recombinant protein biochemistry, cell culture models, and mutant knock-in mice were used to study pathophysiologic mechanisms.

Results

Two kindred were identified with autosomal dominant behavioral variant frontotemporal dementia. Neuropathologic and radiologic studies indicated that neurodegeneration was associated with neurofibrillary tangles. In addition, there was abnormal vacuolization of neocortical neurons. These "Vacuolar Tauopathy" cases were assocaited with a novel VCP (Vaolsin-Containing Protein) mutation, distinct from mutations that cause TDP-43 proteinopathy. Recombinant protein biochemistry demonstrated that the vacuolar tauopathy mutation was associated with a partial loss of function. Moreover, recombinant VCP appeared to exhibit disaggregase activity against pathologic tau and mutant VCP expression was associated with enhanced tau aggregation in both cellular and animal models. Ongoing studies suggest that there are mechanistic differences between VCP mutations that cause tauopathy versus TDP-43 proteinopathy.

Conclusions

Vacuolar Tauopathy is a novel autosomal dominant tauopathy associated with a hypomorph VCP mutation which impairs disaggregase activity. Different mutations of the same gene (VCP) can lead to different underlying neuropathologies (tau versus TDP-43) but the same clinical presentation (FTD), a remarkable instance of allelic heterogeneity. These results suggest that VCP may be a novel target for the treatment of tauopathies.

Aims

Heterozygous loss of function (LOF) mutations in the GRN gene cause the neurodegenerative disease frontotemporal dementia (GRN-FTD). GRN encodes progranulin (PGRN), a soluble lysosomal and secreted protein, which is highly expressed in microglia and regulates critical lysosomal functions, inflammation, and neuronal survival. Because GRN-FTD patients have reduced levels of PGRN in biofluids and tissues, including brain, we assessed the potential of a CNS-penetrant protein replacement therapy to slow or prevent disease progression.

Methods

Here we describe a novel biotherapeutic termed Protein Transport Vehicle (PTV):PGRN. PTV:PGRN consists of recombinant human PGRN fused to a modified Fc domain engineered to bind to the apical domain of the human transferrin receptor (huTfR), enabling receptor-mediated transcytosis of the drug across the blood-brain barrier and into the brain parenchyma.

Results

In vitro studies showed that treatment with PTV:PGRN fully rescues a range of Grn KO cell phenotypes, including lysosomal proteolysis, endolysosomal phospholipid bis(monoacylglycero)phosphate (BMP) levels, which promotes lysosomal lipid catabolism, and TREM2 levels. Intravenous administration of PTV:PGRN in knock-in (KI) mice expressing huTfR led to significant increase in brain PGRN levels relative to Fc:PGRN fusion control, highlighting the ability of huTfR binding to increase brain uptake of PGRN. Systemic administration of PTV:PGRN corrected disease-associated lysosomal phenotypes, including BMP deficiency and lipofuscinosis, as well as inflammatory markers in the brain of Grn KO x huTfR KI mice.

Conclusions

Our data suggest that PTV:PGRN may represent a viable therapeutic strategy for the treatment of GRN-FTD and potentially other neurological disorders associated with PGRN deficiency.