Screening for chemical modulators for LRRK2

After the discovery of leucine-rich repeat kinase 2 (LRRK2) as a risk factor for sporadic Parkinson's disease (PD) and mutations in LRRK2 as a cause of some forms of familial PD, there has been substantial interest in finding chemical modulators of LRRK2 function. Most of the pathogenic mutations in LRRK2 are within the enzymatic cores of the protein; therefore, many screens have focused on finding chemical modulators of this enzymatic activity. There are alternative screening approaches that could be taken to investigate compounds that modulate LRRK2 cellular functions. These screens are more often phenotypic screens. The preparation for a screen has to be rigorous and enable high-throughput accurate assessment of a compound's activity. The pipeline to beginning a drug screen and some LRRK2 inhibitor and phenotypic screens will be discussed

Drosophila Parkin requires PINK1 for mitochondrial translocation and ubiquitinates Mitofusin

Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson's disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including protein degradation, mitochondrial homeostasis, and autophagy; however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting mitochondrial fission and/or fusion, to mediate their autophagic turnover. The precise mechanism of recruitment and the ubiquitination target are unclear. Here we show in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. We hypothesize that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy

Deficits in mitochondrial spare respiratory capacity contribute to the neuropsychological changes of alzheimer’s disease

Alzheimer’s disease (AD) is diagnosed using neuropsychological testing, supported by amyloid and tau biomarkers and neuroimaging abnormalities. The cause of neuropsychological changes is not clear since they do not correlate with biomarkers. This study investigated if changes in cellular metabolism in AD correlate with neuropsychological changes. Fibroblasts were taken from 10 AD patients and 10 controls. Metabolic assessment included measuring total cellular ATP, extracellular lactate, mitochondrial membrane potential (MMP), mitochondrial respiration and glycolytic function. All participants were assessed with neuropsychological testing and brain structural MRI. AD patients had significantly lower scores in delayed and immediate recall, semantic memory, phonemic fluency and Mini Mental State Examination (MMSE). AD patients also had significantly smaller left hippocampal, left parietal, right parietal and anterior medial prefrontal cortical grey matter volumes. Fibroblast MMP, mitochondrial spare respiratory capacity (MSRC), glycolytic reserve, and extracellular lactate were found to be lower in AD patients. MSRC/MMP correlated significantly with semantic memory, immediate and delayed episodic recall. Correlations between MSRC and delayed episodic recall remained significant after controlling for age, education and brain reserve. Grey matter volumes did not correlate with MRSC/MMP. AD fibroblast metabolic assessment may represent an emergent disease biomarker of AD

Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS)

Background: Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3-10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis. Methodology/Principal Findings: Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p. Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B. Conclusions/Significance: We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS. We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype

Oxidative switch drives mitophagy defects in dopaminergic parkin mutant patient neurons

Mutations in PRKN are the most common cause of early onset Parkinson’s disease. Parkin is an E3 ubiquitin ligase, functioning in mitophagy. Mitochondrial abnormalities are present in PRKN mutant models. Patient derived neurons are a promising model in which to study pathogenic mechanisms and therapeutic targets. Here we generate induced neuronal progenitor cells from PRKN mutant patient fibroblasts with a high dopaminergic neuron yield. We reveal changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Fibroblasts from 4 controls and 4 PRKN mutant patients were transformed into induced neuronal progenitor cells and subsequently differentiated into dopaminergic neurons. Mitochondrial morphology, function and mitophagy were evaluated using live cell fluorescent imaging, cellular ATP and reactive oxygen species production quantification. Direct conversion of control and PRKN mutant patient fibroblasts results in induced neuronal progenitor and their differentiation yields high percentage of dopaminergic neurons. We were able to observe changing mitochondrial phenotypes as neurons undergo a metabolic switch during differentiation. Our results show that when pre-neurons are glycolytic early in differentiation mitophagy is unimpaired by PRKN deficiency. However as neurons become oxidative phosphorylation dependent, mitophagy is severely impaired in the PRKN mutant patient neurons. These changes correlate with changes in mitochondrial function and morphology; resulting in lower neuron yield and altered neuronal morphology. Induced neuronal progenitor cell conversion can produce a high yield of dopaminergic neurons. The mitochondrial phenotype, including mitophagy status, is highly dependent on the metabolic status of the cell. Only when neurons are oxidative phosphorylation reliant the extent of mitochondrial abnormalities are identified. These data provide insight into cell specific effects of PRKN mutations, in particular in relation to mitophagy dependent disease phenotypes and provide avenues for alternative therapeutic approaches

Inhibition of the mitochondrial calcium uniporter (MCU) rescues dopaminergic neurons in pink1-/- zebrafish

Mutations in PTEN-induced putative kinase 1 (PINK1) are a cause of early onset Parkinson's disease (PD). Loss of PINK1 function causes dysregulation of mitochondrial calcium homeostasis, resulting in mitochondrial dysfunction and neuronal cell death. We report that both genetic and pharmacological inactivation of the mitochondrial calcium uniporter (MCU), located in the inner mitochondrial membrane, prevents dopaminergic neuronal cell loss in pink1Y431* mutant zebrafish (Danio rerio) via rescue of mitochondrial respiratory chain function. In contrast, genetic inactivation of the voltage dependent anion channel 1 (VDAC1), located in the outer mitochondrial membrane, did not rescue dopaminergic neurons in PINK1 deficient Danio rerio. Subsequent gene expression studies revealed specific upregulation of the mcu regulator micu1 in pink1Y431* mutant zebrafish larvae and inactivation of micu1 also results in rescue of dopaminergic neurons. The functional consequences of PINK1 deficiency and modified MCU activity were confirmed using a dynamic in silico model of Ca2+ triggered mitochondrial activity. Our data suggest modulation of MCU-mediated mitochondrial calcium homeostasis as a possible neuroprotective strategy in PINK1 mutant PD

Mitochondrial dynamics–fusion, fission, movement, and mitophagy–in neurodegenerative diseases

Neurons are metabolically active cells with high energy demands at locations distant from the cell body. As a result, these cells are particularly dependent on mitochondrial function, as reflected by the observation that diseases of mitochondrial dysfunction often have a neurodegenerative component. Recent discoveries have highlighted that neurons are reliant particularly on the dynamic properties of mitochondria. Mitochondria are dynamic organelles by several criteria. They engage in repeated cycles of fusion and fission, which serve to intermix the lipids and contents of a population of mitochondria. In addition, mitochondria are actively recruited to subcellular sites, such as the axonal and dendritic processes of neurons. Finally, the quality of a mitochondrial population is maintained through mitophagy, a form of autophagy in which defective mitochondria are selectively degraded. We review the general features of mitochondrial dynamics, incorporating recent findings on mitochondrial fusion, fission, transport and mitophagy. Defects in these key features are associated with neurodegenerative disease. Charcot-Marie-Tooth type 2A, a peripheral neuropathy, and dominant optic atrophy, an inherited optic neuropathy, result from a primary deficiency of mitochondrial fusion. Moreover, several major neurodegenerative diseases—including Parkinson's, Alzheimer's and Huntington's disease—involve disruption of mitochondrial dynamics. Remarkably, in several disease models, the manipulation of mitochondrial fusion or fission can partially rescue disease phenotypes. We review how mitochondrial dynamics is altered in these neurodegenerative diseases and discuss the reciprocal interactions between mitochondrial fusion, fission, transport and mitophagy

Rescue of mitochondrial function in parkin-mutant Fibroblasts using drug loaded PMPC-PDPA polymersomes and tubular polymersomes

Mutations in parkin cause autosomal recessive Parkinsonism and mitochondrial defects. A recent drug screen identified a class of steroid-like hydrophobic compounds able to rescue mitochondrial function in parkin-mutant fibroblasts. Whilst these possess therapeutic potential, the size and high hydrophobicity of some may limit their ability to penetrate the blood-brain barrier from systemic circulation, something that could be improved by novel drug formulations. In the present study, the steroid-like compounds Ursolic Acid (UA) and Ursocholanic Acid (UCA) were successfully encapsulated within nanoscopic polymersomes formed by poly(2-(methacryloyloxy)ethyl phosphorylcholine)–poly(2-di-isopropylamino)ethyl methacrylate) (PMPC-PDPA) and separated into spherical and tubular morphologies to assess the effects of nanoparticle mediated delivery on drug efficacy. Following incubation with either morphology, parkin-mutant fibroblasts demonstrated time and concentration dependent increases in intracellular ATP levels, resembling those resulting from treatment with nascent UA and UCA formulated in 0.1% DMSO, as used in the original drug screen. Empty PMPC-PDPA polymersomes did not alter physiological measures related to mitochondrial function or induce cytotoxicity. In combination with other techniques such as ligand functionalisation, PMPC-PDPA nanoparticles of well-defined morphology may prove a promising platform for tailoring the pharmacokinetic profile and organ specific bio-distribution of highly hydrophobic compounds

What influences students in their development of socio-emotional intelligence whilst at university?

This qualitative study undertaken at a University in England investigates what influences the development of undergraduate students’ socio-emotional intelligence (SEI). Through a thematic analysis of semi-structured interviews with students and lecturers, the study highlights various approaches that the learning environment, both physical and cultural influenced their development of SEI. Learning in small groups where students felt safe and supported impacted on their sense of self, and helped develop their confidence. Reflecting on their own experience without constraints or assessed outcomes was also beneficial to these students. The lecturer’s knowledge and expertise, and how they modelled SEI were seen as imperative and meaningful to the development of students’ SEI. This paper concludes that students must be supported to develop holistically, integrating cognition and emotion, making practical suggestions as to how this may be achieved. Given the paucity of research in this area, opportunities for further research are highlighte

Moving lessons: teaching sociology through embodied learning in the HE classroom

This chapter outlines an approach to classroom teaching that makes use of physical movement alongside more traditional lecturing methods when delivering lessons on abstract theoretical material. It develops the notion of embodied learning as a 'physical metaphor', outlining some examples of this practice that we have used in our recent work with a class of first year undergraduates. We argue that conceptualising students as embodied subjects, whose capacity to learn extends through and beyond their physical selves, educators are able to enhance classroom delivery by diversifying teaching activities and creating opportunities for enjoyable and memorable learning experiences. We advocate the reflexive, contextually-sensitive and level- appropriate use of this method, arguing that despite some limitations it can animate students' understanding of academic ideas in uniquely personalised ways