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2.  Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation 
Scientific Reports  2016;6:29200.
Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer’s disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes.
doi:10.1038/srep29200
PMCID: PMC4935877  PMID: 27383650
3.  Tau Protein Hyperphosphorylation and Aggregation in Alzheimer’s Disease and Other Tauopathies, and Possible Neuroprotective Strategies 
Biomolecules  2016;6(1):6.
Abnormal deposition of misprocessed and aggregated proteins is a common final pathway of most neurodegenerative diseases, including Alzheimer’s disease (AD). AD is characterized by the extraneuronal deposition of the amyloid β (Aβ) protein in the form of plaques and the intraneuronal aggregation of the microtubule-associated protein tau in the form of filaments. Based on the biochemically diverse range of pathological tau proteins, a number of approaches have been proposed to develop new potential therapeutics. Here we discuss some of the most promising ones: inhibition of tau phosphorylation, proteolysis and aggregation, promotion of intra- and extracellular tau clearance, and stabilization of microtubules. We also emphasize the need to achieve a full understanding of the biological roles and post-translational modifications of normal tau, as well as the molecular events responsible for selective neuronal vulnerability to tau pathology and its propagation. It is concluded that answering key questions on the relationship between Aβ and tau pathology should lead to a better understanding of the nature of secondary tauopathies, especially AD, and open new therapeutic targets and strategies.
doi:10.3390/biom6010006
PMCID: PMC4808800  PMID: 26751493
Alzheimer’s disease; amyloid β; neurofibrillary degeneration; microtubules; neuropathology; phosphorylation; protein aggregation; protein oligomerization; tauopathies; tau protein
4.  Using human induced pluripotent stem cells to model cerebellar disease: Hope and hype 
Journal of Neurogenetics  2015;29(2-3):95-102.
The cerebellum forms a highly ordered and indispensible component of motor function within the adult neuraxis, consisting of several distinct cellular subtypes. Cerebellar disease, through a variety of genetic and acquired causes, results in the loss of function of defined subclasses of neurons, and remains a significant and untreatable health care burden. The scarcity of therapies in this arena can partially be explained by unresolved disease mechanisms due to inaccessibility of human cerebellar neurons in a relevant experimental context where initiating disease mechanisms could be functionally elucidated, or drug screens conducted. In this review we discuss the potential promise of human induced pluripotent stem cells (hiPSCs) for regenerative neurology, with a particular emphasis on in vitro modelling of cerebellar degeneration. We discuss progress made thus far using hiPSC-based models of neurodegeneration, noting the relatively slower pace of discovery made in modelling cerebellar dysfunction. We conclude by speculating how strategies attempting cerebellar differentiation from hiPSCs can be refined to allow the generation of accurate disease models. This in turn will permit a greater understanding of cerebellar pathophysiology to inform mechanistically rationalised therapies, which are desperately needed in this field.
doi:10.3109/01677063.2015.1053478
PMCID: PMC4673530  PMID: 25985846
Cerebellum; directed differentiation; disease modelling; iPSC-technology; reprogramming
5.  Developmental regulation of tau splicing is disrupted in stem cell-derived neurons from frontotemporal dementia patients with the 10 + 16 splice-site mutation in MAPT 
Human Molecular Genetics  2015;24(18):5260-5269.
The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC–neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing.
doi:10.1093/hmg/ddv246
PMCID: PMC4550814  PMID: 26136155
6.  Screening a UK amyotrophic lateral sclerosis cohort provides evidence of multiple origins of the C9orf72 expansion☆ 
Neurobiology of Aging  2015;36(1):546.e1-546.e7.
An expanded hexanucleotide repeat in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD). Although 0–30 hexanucleotide repeats are present in the general population, expansions >500 repeats are associated with C9ALS/FTD. Large C9ALS/FTD expansions share a common haplotype and whether these expansions derive from a single founder or occur more frequently on a predisposing haplotype is yet to be determined and is relevant to disease pathomechanisms. Furthermore, although cases carrying 50–200 repeats have been described, their role and the pathogenic threshold of the expansions remain to be identified and carry importance for diagnostics and genetic counseling. We present clinical and genetic data from a UK ALS cohort and report the detailed molecular study of an atypical somatically unstable expansion of 90 repeats. Our results across different tissues provide evidence for the pathogenicity of this repeat number by showing they can somatically expand in the central nervous system to the well characterized pathogenic range. Our results support the occurrence of multiple expansion events for C9ALS/FTD.
doi:10.1016/j.neurobiolaging.2014.07.037
PMCID: PMC4270445  PMID: 25179228
Frontotemporal dementia; Somatic instability; Amyotrophic lateral sclerosis
7.  The Parkinson’s disease genes Fbxo7 and Parkin interact to mediate mitophagy 
Nature neuroscience  2013;16(9):10.1038/nn.3489.
Compelling evidence indicates that two autosomal recessive Parkinson’s disease genes, PINK1 (PARK6) and Parkin (PARK2), co-operate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain containing protein Fbxo7 (PARK15) also cause early onset autosomal recessive Parkinson’s disease by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and plays a role in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression show deficiencies in Parkin mitochondrial translocation, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin supporting a functional relationship between the two proteins. Parkinson’s disease-causing mutations in Fbxo7 interfere with this process, emphasising the importance of mitochondrial dysfunction in Parkinson’s disease pathogenesis.
doi:10.1038/nn.3489
PMCID: PMC3827746  PMID: 23933751
Fbxo7; Parkin; PINK1; mitofusin 1; mitophagy; Drosophila; Parkinson’s disease
8.  Calpain cleavage and inactivation of the sodium calcium exchanger-3 occur downstream of Aβ in Alzheimer’s disease 
Aging Cell  2013;13(1):49-59.
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by pathological deposits of β-amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase-3, activity is a prominent feature of AD brain. In addition, we observe increased calpain-mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aβ1–42. We also show that exposure of primary cortical neurons to oligomeric Aβ1–42 results in calpain-dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aβ toxicity. Our findings suggest that Aβ mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.
doi:10.1111/acel.12148
PMCID: PMC4326873  PMID: 23919677
Alzheimer’s disease; beta-amyloid; calcium; calpain; sodium calcium exchanger; tau
9.  Pathogenic VCP Mutations Induce Mitochondrial Uncoupling and Reduced ATP Levels 
Neuron  2013;78(1):57-64.
Summary
Valosin-containing protein (VCP) is a highly expressed member of the type II AAA+ ATPase family. VCP mutations are the cause of inclusion body myopathy, Paget’s disease of the bone, and frontotemporal dementia (IBMPFD) and they account for 1%–2% of familial amyotrophic lateral sclerosis (ALS). Using fibroblasts from patients carrying three independent pathogenic mutations in the VCP gene, we show that VCP deficiency causes profound mitochondrial uncoupling leading to decreased mitochondrial membrane potential and increased mitochondrial oxygen consumption. This mitochondrial uncoupling results in a significant reduction of cellular ATP production. Decreased ATP levels in VCP-deficient cells lower their energy capacity, making them more vulnerable to high energy-demanding processes such as ischemia. Our findings propose a mechanism by which pathogenic VCP mutations lead to cell death.
Highlights
► VCP deficiency is associated with mitochondrial depolarization ► VCP deficiency leads to increased mitochondrial respiration and uncoupling ► ATP levels are decreased in VCP-deficient cells due to lower ATP production
In this study, Bartolome et al. show that three independent pathogenic VCP mutations induce mitochondrial uncoupling, resulting in low cellular ATP production, rendering the cells more susceptible to cell death under stress-induced ischemic conditions.
doi:10.1016/j.neuron.2013.02.028
PMCID: PMC3843114  PMID: 23498975
10.  Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research 
PLoS ONE  2012;7(8):e43099.
Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.
doi:10.1371/journal.pone.0043099
PMCID: PMC3428297  PMID: 22952635
11.  MAPT expression and splicing is differentially regulated by brain region: relation to genotype and implication for tauopathies 
Human Molecular Genetics  2012;21(18):4094-4103.
The MAPT (microtubule-associated protein tau) locus is one of the most remarkable in neurogenetics due not only to its involvement in multiple neurodegenerative disorders, including progressive supranuclear palsy, corticobasal degeneration, Parksinson's disease and possibly Alzheimer's disease, but also due its genetic evolution and complex alternative splicing features which are, to some extent, linked and so all the more intriguing. Therefore, obtaining robust information regarding the expression, splicing and genetic regulation of this gene within the human brain is of immense importance. In this study, we used 2011 brain samples originating from 439 individuals to provide the most reliable and coherent information on the regional expression, splicing and regulation of MAPT available to date. We found significant regional variation in mRNA expression and splicing of MAPT within the human brain. Furthermore, at the gene level, the regional distribution of mRNA expression and total tau protein expression levels were largely in agreement, appearing to be highly correlated. Finally and most importantly, we show that while the reported H1/H2 association with gene level expression is likely to be due to a technical artefact, this polymorphism is associated with the expression of exon 3-containing isoforms in human brain. These findings would suggest that contrary to the prevailing view, genetic risk factors for neurodegenerative diseases at the MAPT locus are likely to operate by changing mRNA splicing in different brain regions, as opposed to the overall expression of the MAPT gene.
doi:10.1093/hmg/dds238
PMCID: PMC3428157  PMID: 22723018
12.  A Tangled Web – Tau and Sporadic Parkinson's Disease 
Parkinson's disease (PD) represents a major challenge for health care systems around the world: it is the most common degenerative movement disorder of old age, affecting over 100,000 people in the UK alone (Schrag et al., 2000). Despite the remarkable success of treatments directed at potentiating or replacing dopamine within the brain, which can relieve symptoms for over a decade, PD remains an incurable and invariably fatal disorder. As such, efforts to understand the processes that lead to cell death in the brains of patients with PD are a priority for neurodegenerative researchers. A great deal of progress has been made in this regard by taking advantage of advances in genetics, initially by the identification of genes responsible for rare Mendelian forms of PD (outlined in Table 1), and more recently by applying genome wide association studies (GWAS) to the sporadic form of the disease (Hardy et al., 2009). Several such GWAS have now been carried out, with a meta-analysis currently under way. Using over 6000 cases and 10,000 controls, two of these studies have identified variation at a number of loci as being associated with an increased risk of disease (Satake et al., 2009; Simon-Sanchez et al., 2009). Three genes stand out as candidates from these studies – the SNCA gene, coding for α-synuclein, the LRRK2 gene, coding for leucine rich repeat kinase 2, and MAPT, coding for the microtubule-associated protein tau. Mutations at all three of these loci have been associated with Mendelian forms of disease presenting with the clinical syndrome of Parkinsonism, however only SNCA and LRRK2 have been previously associated with pathologically defined PD (Hardy et al., 2009). Point mutations in α-synuclein, along with gene multiplication events, result in autosomal dominant PD, often with a significant dementia component. In addition to this, α-synuclein is the principle component of the main pathological hallmark of idiopathic PD, the Lewy body, making it an unsurprising hit in the GWAS (Spillantini et al., 1997). Mutations in LRRK2 are the most common genetic cause of PD, and so again made this gene a likely candidate as a susceptibility locus for the sporadic form of disease (Kumari and Tan, 2009). More surprising, perhaps, was the identification of tau as a susceptibility factor for Parkinson's. In this review we will outline the role of tau in neurodegeneration and in different forms of Parkinsonism, and speculate as to what the functional basis of the association between MAPT and PD might be.
doi:10.3389/fpsyt.2010.00150
PMCID: PMC3059613  PMID: 21423457
genome wide association study; Parkinson's disease; synuclein; tau

Results 1-12 (12)