The Dynamics of Mitochondrial Transport in Neurons

Department of Biological SciencesMitochondria are an essential component of axons, and proper distribution and transport of these organelles is critical for the maintenance of neuronal health. These organelles are responsible for oxidative phosphorylation, the primary energy metabolic pathway in neurons, as well as buffering of calcium micro-spikes that are a consequence of neuronal signaling. While mitochondria are present in the majority of eukaryotic cell types, their positioning is especially important in axons due to the significant length of these extensions, and potential distance from the cell body. Mitochondria in axons are actively transported anterograde and retrograde relative to the cell body, and disruption of this mechanism is known to be responsible for a variety of neurological diseases. The molecular aspects regulating mitochondrial transport have been well studiedhowever, there has been comparatively little focus on the large-scale aspects of this phenomenon, across the whole axon. Certain assumptions regarding the overall destination and purpose of mitochondrial transport are based upon high-resolution, low field of view microscopy studies, and do not take into account the dynamics of the entire axon. In this study, we have developed a new method for examining whole-axon movement over significant periods of time. This was done through the use of mitochondria-localized dendra2, a photoconvertible protein that is able to switch from green fluorescence to red fluorescence through a brief exposure to 405 nm laser radiation. By converting mito-dendra2 in either the proximal (soma) or distal areas of the axon, we were able to track individual mitochondria over long periods and distances using low resolution and high field of view microscopy techniques. The resulting data could be used to described total mobility of mitochondria moving in a particular direction, as well as the distribution of stationary mitochondria. We were also able to further characterize the nature of mobile mitochondria by quantifying their movement into a velocity distribution. This distribution could be fitted to a derivation of the Fokker-Planck equation, which examines the nature of movement of particles through a given space. Using this new protocol, we were able to find significant differences between anterograde and retrograde movement. This protocol was also sensitive enough to detect differences in velocity when the mitochondria specific E3 ubiquitin ligase, Parkin, was overexpressed, showing that this method is a useful application for examining elements that perturb mitochondrial movement. Using this new methodology, we have also examined the role that mitochondrial calcium uniporter (MCU), an inner mitochondrial membrane pore-forming unit, plays in mitochondrial movement. MCU has been previously implicated in mitochondrial mobility regulation through the use of pharmaceutical methods, though a specific biochemical relationship between the mitochondria transport components and MCU has not been established. Here, we have found that reduction in MCU levels in axons significantly reduces both the total number, as well as the velocity, of moving mitochondria. Biochemical analysis reveals that this is due to an interaction with Miro1, the primary regulator of mitochondrial transport. While Miro1 and MCU are outer and inner mitochondrial membrane proteins, respectively, MCU is able to bind to Miro1 through its previously defined mitochondrial targeting sequence (MTS). We found that MCU???s MTS is localized to the mitochondrial outer membrane, and is dispensable for localization of MCU to mitochondria. This interaction is vital for mitochondrial transport to occur, and overexpression of an MCU mutant without the MTS is unable to rescue transport defects caused by MCU knockdown. Altogether, this body of work explores multiple aspects of mitochondrial transport in axons, and reveals levels of complexity not previously described.ope

Miro, MCU, and calcium: Bridging our understanding of mitochondrial movement in axons

Neurons are extremely polarized structures with long axons and dendrites, which require proper distribution of mitochondria and maintenance of mitochondrial dynamics for neuronal functions and survival. Indeed, recent studies show that various neurological disorders are linked to mitochondrial transport in neurons. Mitochondrial anterograde transport is believed to deliver metabolic energy to synaptic terminals where energy demands are high, while mitochondrial retrograde transport is required to repair or remove damaged mitochondria in axons. It has been suggested that Ca2+ plays a key role in regulating mitochondrial transport by altering the configuration of mitochondrial protein, miro. However, molecular mechanisms that regulate mitochondrial transport in neurons still are not well characterized. In this review, we will discuss the roles of miro in mitochondrial transport and how the recently identified components of the mitochondrial calcium uniporter add to our current model of mitochondrial mobility regulation.open2

Dynamics of mitochondrial transport in axons

The polarized structure and long neurites of neurons pose a unique challenge for proper mitochondrial distribution. It is widely accepted that mitochondria move from the cell body to axon ends and vice versa; however, we have found that mitochondria originating from the axon ends moving in the retrograde direction never reach to the cell body, and only a limited number of mitochondria moving in the anterograde direction from the cell body arrive at the axon ends of mouse hippocampal neurons. Furthermore, we have derived a mathematical formula using the Fokker-Planck equation to characterize features of mitochondrial transport, and the equation could determine altered mitochondrial transport in axons overexpressing parkin. Our analysis will provide new insights into the dynamics of mitochondrial transport in axons of normal and unhealthy neurons.clos

Séreignarsparnaður og fyrstu kaup

Þessi ritgerð fjallar um þekkingu ungs fólks á notagildi séreignarsparnaðar. Þar að auki er fjallað um hvernig séreignarsparnaður getur verið notaður til að greiða fyrstu kaup og niðurgreiða húsnæðislán. Séreignarsparnaður er tekinn fyrir, greint er frá séreignarsparnaðinum sem sparileið og útskýrt er hvernig úrræðið getur verið nýtt inn á húsnæðislán. Spurningakönnun var lögð fram fyrir almenningi og kom í ljós að flestir þekkja til séreignarsparnaðar en einnig þyrfti betri fræðslu til þess að bæta þekkingu á notagildi sparnaðarleiðarinnar. Fjallað er um mismunandi þætti sem hafa áhrif á notkun séreignarsparnaðar meðal ungs fólks. Niðurstöður leiddu í ljós að þau sem höfðu fengið fræðslu um sparileiðina voru líklegri til þess að nýta sér hana. Önnur áhugaverð niðurstaða var að helmingur þeirra sem þekkja til séreignarsparnaðarins hafa heyrt um möguleikann að nýta sér hann til fyrstu húsnæðiskaupa. Sú aðferð aðstoðar ungt fólk við að kaupa sér sína fyrstu eign, til hið minnsta brúar bilið fyrir þessi miklu og flóknu kaup. Ályktunin er sú að það sé ábótavant að bæta fræðslu um séreignarsparnað til þess að ungt fólk geti nýtt sér þessa sparnaðarleið á sem bestan hátt. Með öflugri fræðslu er hægt að bæta þekkingu ungs fólks á sparnaðarleiðinni og þar að leiðandi auðvelda þeim að eignast þeirra fyrstu eign fyrr. Þau sitja þá uppi með fleiri tól og aukna aðstoð við að ná stórum sparnaðarmarkmiðum í framtíðinni

Home reading to meet the needs of struggling readers : training parents to implement systematic phonics instruction

Algengasta heimavinnan á yngsta stigi í grunnskólum hérlendis er heimalestur. Fátt er um íslenskar rannsóknir á framkvæmd og áhrifum slíkrar lestrarþjálfunar. Erlendar rannsóknir benda til að foreldrar geti haft töluverð jákvæð áhrif á lestrarnám barna sinna með því að leggja fyrir æfingar sem byggja upp grundvallarfærni í lestri, svo sem hljóðun, umskráningu og lesfimi. Meðal þess sem einkennir gæða lestrarþjálfun og eykur líkur á framförum barna í lestrarvanda eru mörg tækifæri til svörunar (e. opportunities to respond), tíðni jákvæðrar endurgjafar, og skjót leiðrétting villna. G-PALS (e. First Grade Peer-Assisted Learning Strategies) er kennsluaðferð sem felur í sér markvissa hljóðaaðferð, fjölda tækifæra til svörunar, tíða jákvæða endurgjöf og villuleiðréttingu. Markmið með rannsókn þessari var að kanna áhrif þess að kenna foreldrum að nota markvissa hljóðaaðferð í formi G-PALS verkefna á gæði heimalestrarstunda og færni barna í hljóðun bókstafa, umskráningu, lestri orða og lesfimi. Þátttakendur í rannsókninni voru þrír drengir og tvær stúlkur í 3.bekk í stórum grunnskóla á höfuðborgarsvæðinu ásamt foreldrum þeirra. Nemendurnir voru tilnefndir til þátttöku af umsjónarkennurum sínum þar sem lesfimi þeirra hafði mælst undir eða í kringum lægsta viðmiðið í Lesferli Menntamálastofnunar í janúar í 3.bekk, rétt fyrir upphaf rannsóknar. Einn nemandi hafði íslensku sem annað tungumál, einn var greindur með mótþróaþrjóskuröskun og tveir með athyglisbrest með/án ofvirkni. Staða nemenda í lestrarnáminu var metin með mælingum á hljóðaþekkingu, hljóðafimi, orðleysufimi og fimi í lestri algengra orða. Auk þess voru útbúin G-PALS kaflapróf sem voru notuð til mælinga á hljóða- og orðafimi í G-PALS námsefni. Foreldrar tóku heimalestrarstundir upp á hljóðupptökur og deildu þeim með rannsakanda. Við upphaf inngripsskeiðs fengu foreldrar stutt námskeið sem m.a. fól í sér myndskeið þar sem framkvæmd G-PALS verkefnis var lýst skref fyrir skref. Eftir það voru vikulegir handleiðslufundir þar sem foreldrar fengu endurgjöf á framkvæmd G-PALS í heimalestri. G-PALS verkefni voru valin í samræmi við lestrarfærnimælingar hvers nemanda og sendar heim með nemendum að jafnaði einu sinni í viku. Margfalt grunnskeiðssnið yfir þátttakendur sýndi að með notkun G-PALS lengdust heimalestrarstundir, tækifærum til svörunar fjölgaði, tíðni jákvæðrar endurgjafar jókst og villum í lestri fækkaði. Hlutfall villna sem voru leiðréttar í heimalestri hækkaði hjá einni þátttakendafjölskyldu. Heimalestur með G-PALS á tveggja til sjö vikna tímabili hafði talsverð jákvæð áhrif á hljóðafimi nemenda en lítil áhrif á hljóðaþekkingu og orðleysufimi. Hljóða- og orðafimi í G-PALS námsefni jókst hjá öllum þátttakendum. Niðurstöður benda til þess að notkun G-PALS námsefnis gæti gagnast við að sníða heimalestur betur að þörfum barna og aðstoða foreldra við að auka gæði heimalestrarstunda. Þörf er á frekari rannsóknum til að meta áhrif mismunandi heimalestrarþjálfunar á lestrarfærni nemenda.The purpose of this study was to investigate the effect of teaching parents to use Systematic Phonics Instruction in the form of First Grade PALS on the quality of home reading and children‘s letter sound recognition, letter sound fluency, word and non-word reading fluency. The participants were three boys and two girls in 3rd grade in a large elementary school in the capital area of Iceland, together with their parents. The students were selected by their supervisory teacher as their reading skills had been measured below or around the lowest standard in Lesferill reading fluency test in January in 3rd grade, just before the start of the study. One student had Icelandic as a second language, one was diagnosed with oppositional defiant disorder and two with Attention-deficit/hyperactivity disorder. Parents recorded home reading sessions and shared audio recordings with the researcher. At the beginning of the intervention period, the parents took a short course that included a video explaining the implementation of a First Grade PALS lesson. After that, parents received feedback on their implementation of First Grade PALS in weekly meetings with the researcher. A multiple baseline across participants was implemented. The results showed that the use of First Grade PALS in home reading increased the duration of reading sessions, the number of opportunities to respond and the frequency of positive feedback and, reduced the number of errors in reading. The percentage of errors corrected increased in one out of five participating families of this study. Home reading with First Grade PALS over a period of two to seven weeks had a positive effect on students‘ letter sound fluency, but little effect on letter sound recognition and non-word reading fluency. First Grade PALS curriculum based measures showed an increase in letter sound fluency and word reading fluency. Results indicate that the implementation of First Grade PALS could be useful in tailoring home reading to children‘s needs and helping parents to increase the quality of home reading lessons

MCU Interacts with Mirol to Modulate Mitochondrial Functions In Neurons

Mitochondrial Ca2+ uptake is gated by the mitochondrial calcium uniplex, which is comprised of mitochondrial calcium uniporter (MCU), the Ca2+ pore-forming subunit of the complex, and its regulators. Ca2+ influx through MCU affects both mitochondrial function and movement in neurons, but its direct role in mitochondrial movement has not been explored. In this report, we show a link between MCU and Miro1, a membrane protein known to regulate mitochondrial movement. We find that MCU interacts with Miro1 through MCU's N-terminal domain, previously thought to be the mitochondrial targeting sequence. Our results show that the N-terminus of MCU has a transmembrane domain that traverses the outer mitochondrial membrane, which is dispensable for MCU localization into mitochondria. However, this domain is required for Miro1 interaction and is critical for Miro1 directed movement. Together, our findings reveal Miro1 as a new component of the MCU complex, and that MCU is an important regulator of mitochondrial transport

The Effect of Direct Instruction and Fluency Training on the Reading Skills of Two Ten Year Old Boys

Stýrð kennsla Engelmanns (e. Direct Instruction) er raunprófuð aðferð sem hefur verið árangursrík til kennslu á ýmis konar færni svo sem lestri. Hún felur í sér vel skipulögð verkefni og notkun á handriti með skýrum fyrirmælum fyrir kennara. Fimiþjálfun (e. Precision Teaching) er kennslu- og matstæki sem oft er notað samhliða stýrðri kennslu. Helsta markmið fimiþjálfunar er að fá nemendur til að ná hámarksfærni í því námsefni sem verið er að læra. Táknstyrkjakerfi eru notuð til að ýta undir æskilega hegðun, stuðla að þægilegu lærdómsumhverfi og hvetja fólk áfram. Markmið rannsóknarinnar var að kanna áhrif stýrðrar kennslu og fimiþjálfunar ásamt táknstyrkjakerfis á lestrarfærni tveggja 10 ára drengja. Þátttakendur áttu það sameiginlegt að eiga í erfiðleikum með lestur og höfðu slaka lestrarkunnáttu miðað við aldur. Þeir voru þjálfaðir í hljóðun lágstafa íslenska stafrófsins auk tvíhljóðanna au, ei og ey. Niðurstöður fengust með mælingum sem voru notaðar til að meta frammistöðu þátttakenda og árangur kennslunnar. Báðir þátttakendur sýndu aukna færni í hljóðun lágstafa og lestri tveggja, þriggja og fjögurra stafa orða og orðleysa

High-throughput screening of human induced pluripotent stem cell-derived brain organoids

[Display omitted] •Organoid models can be optimized for use in high-throughput screens to study disease relevant phenotypes.•High-throughput imaging can be used for morphological analyses in neural organoids.•MEA recordings are multiplexed with high-throughput imaging to analyze neuronal circuits at network and single-cell levels. The need for scalable high-throughput screening (HTS) approaches for 3D human stem cell platforms remains a central challenge for disease modeling and drug discovery. We have developed a workflow to screen cortical organoids across platforms. We used serum-free embryoid bodies (SFEBs) derived from human induced pluripotent stem cells (hiPSCs) and employed high-content imaging (HCI) to assess neurite outgrowth and cellular composition within SFEBs. We multiplexed this screening assay with both multi-electrode arrays (MEAs) and single-cell calcium imaging. HCI was used to assess the number of excitatory neurons (VGlut+) in experimental replicates of hiPSC-derived SFEBs, demonstrating experiment-to-experiment consistency. Neurite detection using HCI was applied to assess neurite morphology. MEA analysis showed that firing and burst rates in SFEBs decreased with blockade of NMDARs and AMPARs and increased with GABAR blockade. We also demonstrate effective combination of both MEA and HCI to analyze VGlut+ populations surrounding electrodes within MEAs. HCI-based (Ca2+) transient analysis revealed firing in individual cells surrounding active MEA electrodes. Current methods to generate neural organoids show high degrees of variability, and often require sectioning or special handling for analysis. The protocol outlined in this manuscript generates SFEBs with high degree of consistency making them amenable to complex assays combining HTS and electrophysiology allowing for an in-depth, unbiased analysis. SFEBs can be used in combination with HTS to compensate for experimental variability common in 3D cultures, while significantly decreasing processing speed, making this an efficient starting point for phenotypic drug screening