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Age‐related macular degeneration (AMD) is a multifactorial disease, which is characterized by loss of central vision, affecting one in three people by the age of 75. The Y402H polymorphism in the complement factor H (CFH) gene significantly increases the risk of AMD. We show that Y402H‐AMD‐patient‐specific retinal pigment epithelium (RPE) cells are characterized by a significant reduction in the number of melanosomes, an increased number of swollen lysosome‐like‐vesicles with fragile membranes, Cathepsin D leakage into drusen‐like deposits and reduced lysosomal function. The turnover of C3 is increased significantly in high‐risk RPE cells, resulting in higher internalization and deposition of the terminal complement complex C5b‐9 at the lysosomes. Inhibition of C3 processing via the compstatin analogue Cp40 reverses the disease phenotypes by relieving the lysosomes of their overburden and restoring their function. These findings suggest that modulation of the complement system represents a useful therapeutic approach for AMD patients associated with complement dysregulation., Schematic presentation of autophagy‐lysosomal pathway dysfunction in Y402H‐AMD RPE cells and the impact of Cp40 in restoring the lysosomal function.

This study was funded by the Lewy Body Society and the Michael J. Fox Foundation for Parkinson’s Research. A.H. and B.M.B. gratefully acknowledge funding from the Knut och Alice Wallenberg Stiftelse through the Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden. S.T., M.K.D. and P.D.W. gratefully acknowledge the financial support of the European Regional Development Fund, Scottish Funding Council and Highlands and Islands Enterprise. Newcastle University TEM Services acknowledge BBSRC support (Grant code BB/R013942/1). The Research was supported by the National Institute for Health Research Newcastle Biomedical Research Centre based at Newcastle Hospitals NHS Foundation Trust and Newcastle University. The Newcastle Brain Tissue Resource is supported by grants from the UK Medical Research Council and the Brains for Dementia Research, a joint venture between Alzheimer’s Society and Alzheimer’s Research UK. Mutations in glucocerebrosidase (GBA) are the most prevalent genetic risk factor for Lewy body disorders (LBD)—collectively Parkinson’s disease, Parkinson’s disease dementia and dementia with Lewy bodies. Despite this genetic association, it remains unclear how GBA mutations increase susceptibility to develop LBD. We investigated relationships between LBD-specific glucocerebrosidase deficits, GBA-related pathways, and α-synuclein levels in brain tissue from LBD and controls, with and without GBA mutations. We show that LBD is characterised by altered sphingolipid metabolism with prominent elevation of ceramide species, regardless of GBA mutations. Since extracellular vesicles (EV) could be involved in LBD pathogenesis by spreading disease-linked lipids and proteins, we investigated EV derived from post-mortem cerebrospinal fluid (CSF) and brain tissue from GBA mutation carriers and non-carriers. EV purified from LBD CSF and frontal cortex were heavily loaded with ceramides and neurodegeneration-linked proteins including alpha-synuclein and tau. Our in vitro studies demonstrate that LBD EV constitute a “pathological package” capable of inducing aggregation of wild-type alpha-synuclein, mediated through a combination of alpha-synuclein–ceramide interaction and the presence of pathological forms of alpha-synuclein. Together, our findings indicate that abnormalities in ceramide metabolism are a feature of LBD, constituting a promising source of biomarkers, and that GBA mutations likely accelerate the pathological process occurring in sporadic LBD through endolysosomal deficiency. Publisher PDF

Cell-free mitochondrial DNA (cfmtDNA) is detectable in almost all human body fluids and has been associated with the onset and progression of several complex traits. In-life assessments indicate that reduced cfmtDNA is a feature of neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and multiple sclerosis. However, whether this feature is conserved across all neurodegenerative diseases and how it relates to the neurodegenerative processes remains unclear. In this study, we assessed the levels of ventricular cerebrospinal fluid-cfmtDNA (vCSF-cfmtDNA) in a diverse group of neurodegenerative diseases (NDDs) to determine if the in-life observations of reduced cfmtDNA seen in lumbar CSF translated to the post-mortem ventricular CSF. To investigate further, we compared vCSF-cfmtDNA levels to known protein markers of neurodegeneration, synaptic vesicles and mitochondrial integrity. Our data indicate that reduced vCSF-cfmtDNA is a feature specific to Parkinson’s and appears consistent throughout the disease course. Interestingly, we observed increased vCSF-cfmtDNA in the more neuropathologically severe NDD cases, but no association to protein markers of neurodegeneration, suggesting that vCSF-cfmtDNA release is more complex than mere cellular debris produced following neuronal death. We conclude that vCSF-cfmtDNA is reduced in PD, but not other NDDs, and appears to correlate to pathology. Although its utility as a prognostic biomarker is limited, our data indicate that higher levels of vCSF-cfmtDNA is associated with more severe clinical presentations; suggesting that it is associated with the neurodegenerative process. However, as vCSF-cfmtDNA does not appear to correlate to established indicators of neurodegeneration or indeed indicators of mitochondrial mass, further work to elucidate its exact role is needed.

Dementia with Lewy bodies (DLB) is an age-associated neurodegenerative disorder producing progressive cognitive decline that interferes with normal life and daily activities. Neuropathologically, DLB is characterised by the accumulation of aggregated α-synuclein protein in Lewy bodies and Lewy neurites, similar to Parkinson's disease (PD). Extrapyramidal motor features characteristic of PD, are common in DLB patients, but are not essential for the clinical diagnosis of DLB. Since many PD patients develop dementia as disease progresses, there has been controversy about the separation of DLB from PD dementia (PDD) and consensus reports have put forward guidelines to assist clinicians in the identification and management of both syndromes. Here, we present basic concepts and definitions, based on our current understanding, that should guide the community to address open questions that will, hopefully, lead us towards improved diagnosis and novel therapeutic strategies for DLB and other synucleinopathies. peerReviewed

Lewy body disease (LBD) development is enhanced by mutations in the GBA gene coding for glucocerebrosidase (GCase). The mechanism of this association is thought to involve an abnormal lysosomal system and we therefore sought to evaluate if lysosomal changes contribute to the pathogenesis of idiopathic LBD. Analysis of post-mortem frontal cortex tissue from 7 GBA mutation carriers with LBD, 5 GBA mutation carriers with no signs of neurological disease and human neural stem cells exposed to a GCase inhibitor was used to determine how GBA mutation contributes to LBD. GBA mutation carriers demonstrated a significantly reduced level of GCase protein and enzyme activity and retention of glucocerebrosidase isoforms within the endoplasmic reticulum (ER). This was associated with enhanced expression of the lysosomal markers LAMP1 and LAMP2, though the expression of ATP13A2 and Cathepsin D was reduced, along with the decreased activity of Cathepsin D. The ER unfolded protein response (UPR) regulator BiP/GRP78 was reduced by GBA mutation and this was a general phenomenon in LBD. Despite elevation of GRP94 in LBD, individuals with GBA mutations showed reduced GRP94 expression, suggesting an inadequate UPR. Finally, human neural stem cell cultures showed that inhibition of GCase causes acute reduction of BiP, indicating that the UPR is affected by reduced glucocerebrosidase activity. The results indicate that mutation in GBA leads to additional lysosomal abnormalities, enhanced by an impaired UPR, potentially causing α-synuclein accumulation.

The identification of cell-free circulating mitochondrial DNA (ccf-mtDNA) in early-stage Alzheimer's disease (AD) raised the possibility that the same neurodegenerative effect could be observed in Parkinson's disease (PD). Here, and for the first time, we investigated the role of ccf-mtDNA in PD, identifying a significant reduction of ccf-mtDNA in PD patient cerebrospinal fluid (CSF) when compared to controls. Our data demonstrates that CSF ccf-mtDNA is not only a powerful biomarker for PD, but, given that the effect is also observed in AD, is likely a biomarker for neurodegeneration.

Background Lysosomal dysfunction is thought to be a prominent feature in the pathogenetic events leading to Parkinson’s disease (PD). This view is supported by the evidence that mutations in GBA gene, coding the lysosomal hydrolase β-glucocerebrosidase (GCase), are a common genetic risk factor for PD. Recently, GCase activity has been shown to be decreased in substantia nigra and in cerebrospinal fluid of patients diagnosed with PD or dementia with Lewy Bodies (DLB). Here we measured the activity of GCase and other endo-lysosomal enzymes in different brain regions (frontal cortex, caudate, hippocampus, substantia nigra, cerebellum) from PD (n = 26), DLB (n = 16) and age-matched control (n = 13) subjects, screened for GBA mutations. The relative changes in GCase gene expression in substantia nigra were also quantified by real-time PCR. The role of potential confounders (age, sex and post-mortem delay) was also determined. Findings Substantia nigra showed a high activity level for almost all the lysosomal enzymes assessed. GCase activity was significantly decreased in the caudate (−23%) and substantia nigra (−12%) of the PD group; the same trend was observed in DLB. In both groups, a decrease in GCase mRNA was documented in substantia nigra. No other lysosomal hydrolase defects were determined. Conclusion The high level of lysosomal enzymes activity observed in substantia nigra, together with the selective reduction of GCase in PD and DLB patients, further support the link between lysosomal dysfunction and PD pathogenesis, favoring the possible role of GCase as biomarker of synucleinopathy. Mapping the lysosomal enzyme activities across different brain areas can further contribute to the understanding of the role of lysosomal derangement in PD and other synucleinopathies. Electronic supplementary material The online version of this article (doi:10.1186/s13024-015-0010-2) contains supplementary material, which is available to authorized users.

Importance Progressive external ophthalmoplegia (PEO) is a common feature in adults with mitochondrial (mt) DNA maintenance disorders associated with somatic mtDNA deletions in muscle, yet the causal genetic defect in many patients remains undetermined. Observations Whole-exome sequencing identified a novel, heterozygous p.(Gly671Trp) mutation in theAFG3L2gene encoding an mt protease—previously associated with dominant spinocerebellar ataxia type 28 disease—in a patient with indolent ataxia and PEO. Targeted analysis of a larger, genetically undetermined cohort of patients with PEO with suspected mtDNA maintenance abnormalities identified a second unrelated patient with a similar phenotype and a novel, heterozygous p.(Tyr689His)AFG3L2mutation. Analysis of patient fibroblasts revealed mt fragmentation and decreasedAFG3L2transcript expression. Western blotting of patient fibroblast and muscle showed decreased AFG3L2 protein levels. Conclusions and Relevance Our observations suggest thatAFG3L2mutations are another important cause, albeit rare, of a late-onset ataxic PEO phenotype due to a disturbance of mtDNA maintenance.

Importance: While mutations in glucocerebrosidase (GBA1) are associated with an increased risk for Parkinson disease (PD), it is important to establish whether such mutations are also a common risk factor for other Lewy body disorders.Objective: To establish whether GBA1 mutations are a risk factor for dementia with Lewy bodies (DLB).Design: We compared genotype data on patients and controls from 11 centers. Data concerning demographics, age at onset, disease duration, and clinical and pathological features were collected when available. We conducted pooled analyses using logistic regression to investigate GBA1 mutation carrier status as predicting DLB or PD with dementia status, using common control subjects as a reference group. Random-effects meta-analyses were conducted to account for additional heterogeneity.Setting: Eleven centers from sites around the world performing genotyping.Participants: Seven hundred twenty-one cases met diagnostic criteria for DLB and 151 had PD with dementia. We compared these cases with 1962 controls from the same centers matched for age, sex, and ethnicity.Main Outcome Measures: Frequency of GBA1 mutations in cases and controls.Results: We found a significant association between GBA1 mutation carrier status and DLB, with an odds ratio of 8.28 (95% CI, 4.78-14.88). The odds ratio for PD with dementia was 6.48 (95% CI, 2.53-15.37). The mean age at diagnosis of DLB was earlier in GBA1 mutation carriers than in noncarriers (63.5 vs 68.9 years; P Conclusions and Relevance: Mutations in GBA1 are a significant risk factor for DLB. GBA1 mutations likely play an even larger role in the genetic etiology of DLB than in PD, providing insight into the role of glucocerebrosidase in Lewy body disease.

There is a growing body of evidence linking mitochondrial dysfunction, mediated either through inherited mitochondrial DNA (mtDNA) variation or mitochondrial proteomic deficit, to Parkinson's disease (PD). Yet, despite this, the role of somatic mtDNA point mutations and specifically point-mutational burden in PD is poorly understood. Here, we take advantage of recent technical and methodological advances to examine the role of age-related and acquired mtDNA mutation in the largest study of mtDNA in postmortem PD tissue to date. Our data show that PD patients suffer an increase in mtDNA mutational burden in, but no limited to, the substantia nigra pars compacta when compared to matched controls. This mutational burden appears increased in genes encoding cytochrome c oxidase, supportive of previous protein studies of mitochondrial dysfunction in PD. Accepting experimental limitations, our study confirms the important role of age-related mtDNA point mutation in the etiology of PD, moreover, by analyzing 2 distinct brain regions, we are able to show that PD patient brains are more vulnerable to mtDNA mutation overall.

s, 7 Annual UK Neuromuscular Translational Research Conference, 2014 /Neuromuscular Disorders 24S1 (2014) S7–S27 S15 to the age-dependent manifestation or spontaneous recovery of infantile reversible COX deficiency myopathy. We performed immunohistochemistry and immunoblotting with antibodies against COXVI and COXVII isoforms in mice, in human skeletal muscle of controls and patients in different ages (0–6 months, >1 year, adults) and in human muscle cells. Both in mice and humans, the liver type isoforms gradually decreased, and the heart/muscle-type isoforms increased through development in the first weeks of life, confirming an age-dependent isoform switch. In skeletal muscle of a patient with reversible COX deficiency myopathy we proved the existence of the COXVI and COXVII isoform switch, although we could not confirm an association with the clinical recovery. However, understanding developmental changes of the COX isoforms may have implications for other mitochondrial diseases. P31 Genotypic and phenotypic heterogeneity in adult-onset progressive external ophthalmoplegia (PEO) with mitochondrial DNA instability: a systematic review E.W. Sommerville, P.F. Chinnery, G.S. Gorman, R.W. Taylor. Wellcome Trust Centre for Mitochondrial Research, Cookson Building, Framlington Place, Newcastle University, Newcastle upon Tyne, Tyne and Wear, NE2 4HH, UK; Institute of Genetic Medicine, International Centre for Life, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, UK Background: Progressive external ophthalmoplegia (PEO) is an eye movement disorder characterised by extraocular muscle paresis and muscle restricted mitochondrial DNA (mtDNA) deletions. Patient classification is difficult due to overlapping clinical phenotypes and poor genotype-phenotype correlates. This is compounded by the fact that approximately half of PEO patients do not have a genetic diagnosis. Aims: To review the phenotypic and genotypic manifestations of adult-onset PEO and to identify possible novel candidate genes. Patients: Patients were identified in the literature using electronic searches from Scopus, Medline via PubMed and Genetics Abstracts databases (1 January 1970 to 8 November 2013). Adult patients presenting PEO (≥16 years) presenting with PEO and mtDNA instability and with a confirmed genetic diagnosis were selected. The criterion was extended when searching candidate novel genes. Results: We identified 575 PEO patients, harbouring 12 known nuclear encoded genes (TYMP, SLC25A4, POLG, C10ORF2, OPA1, POLG2, RRM2B, TK2, DGUOK , MPV17, MGME1, and DNA2). Additional novel candidate genes (twenty in total), including several encoding proteins not predicted to localise to mitochondria, were also identified. Conclusion: We propose to use the findings of this systematic review coupled to whole exome and targeted next-generation sequencing technology, to help direct the investigation of a large cohort of clinically well-defined, genetically undetermined adult patients with PEO and mtDNA instability. P32 Mutations in SPG7 cause chronic progressive external ophthalmoplegia through disordered mtDNA maintenance G.S. Gorman, G. Pfeffer, H. Griffin, M. Kurzawa-Akanbi, E.L. Blakely, I. Wilson, K. Sitarz, D. Moore, J.L. Murphy, C.L. Alston, A. Pyle, J. Coxhead, B. Payne, G.H. Gorrie, C. Longman, M. Hadjivassiliou, J. McConville, D. Dick, I. Imam, D. Hilton, F. Norwood, M.R. Baker, S.R. Jaiser, P. Yu-Wai-Man, M. Farrell, A. McCarthy, T. Lynch, R. McFarland, A.M. Schaefer, D.M. Turnbull, R. Horvath, R.W. Taylor, P.F. Chinnery. Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK Background: Despite being a canonical presenting feature of mitochondrial (mt) disease, the genetic basis of progressive external ophthalmoplegia (PEO) remains unknown in a large proportion of patients. Aims: To identify the causative gene in patients with genetically undetermined mtDNA maintenance disorders. Methods: Whole exome sequencing, targeted Sanger sequencing and MLPA analysis were used to study 68 adult patients with PEO either with or without multiple mtDNA deletions in skeletal muscle. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mtDNA analysis and deep re-sequencing of mtDNA. Results: Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), and c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184–3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid adult life with either PEO/ptosis and spastic ataxia, or a progressive ataxic disorder. Functional studies revealed increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mtDNA mutations. Conclusion: The SPG7 gene should be screened in patients in whom a disorder of mtDNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely due to the clonal expansion of secondary mtDNA mutations modulating the phenotype, driven by compensatory mitochondrial biogenesis. a Joint first authors. P33 Do modulators of mitophagy select pathogenic mtDNA mutations? A. Hinks-Roberts, E. Dombi, A. Diot, C. Liao, K. Morten, J. Carver, T. Lodge, H. Mortiboys, J. Poulton. Nuffield Department of Obstetrics and Gynaecology, University of Oxford, UK; Sheffield Institute for Translational Neuroscience, University of Sheffield, UK Mitochondrial diseases that result from maternally transmitted mitochondrial DNA (mtDNA) mutations occur in 1/400 individuals. In heteroplasmic diseases, the balance between co-existing mutant and wild type mtDNA usually underlies disease progression. Cellular mechanisms for maintaining mitochondrial quality include mitophagy, and this could be a critical determinant of disease severity. Previous investigators showed that mitophagy was increased by the drug phenanthroline, a metallopeptidase inhibitor. We reasoned that activating mitophagy with phenanthroline might potentially reduce the load of pathogenic mutant mtDNA in tissue culture cells. We used a previously developed high throughput imaging for quantifying mitophagy in cultured primary fibroblasts bearing the common pathogenic A3243G mtDNA mutation, associated with the mitochondrial encephalomyopathy, lactic acidosis, and strokelike episode syndrome (MELAS) and with diabetes mellitus and deafness. We showed that phenanthroline significantly increased mitophagy in fibroblasts and reduced both the mitochondrial volume and mtDNA content. We conclude that phenanthroline activates mitophagy. However, there was little evidence that it reduced the load of mutant mtDNA. This suggests that both wild type and mutant mtDNA are turned over during mitophagy activated by phenanthroline. We conclude that phenanthroline is a poorly selective activator of mitophagy. Modulators of mitophagy need to target mitochondria enriched for mutant mtDNA if they are to benefit patients with heteroplasmic mtDNA disease.