Neural Conversion and Patterning of Human Pluripotent Stem Cells: A Developmental Perspective.

Since the reprogramming of adult human terminally differentiated somatic cells into induced pluripotent stem cells (hiPSCs) became a reality in 2007, only eight years have passed. Yet over this relatively short period, myriad experiments have revolutionized previous stem cell dogmata. The tremendous promise of hiPSC technology for regenerative medicine has fuelled rising expectations from both the public and scientific communities alike. In order to effectively harness hiPSCs to uncover fundamental mechanisms of disease, it is imperative to first understand the developmental neurobiology underpinning their lineage restriction choices in order to predictably manipulate cell fate to desired derivatives. Significant progress in developmental biology provides an invaluable resource for rationalising directed differentiation of hiPSCs to cellular derivatives of the nervous system. In this paper we begin by reviewing core developmental concepts underlying neural induction in order to provide context for how such insights have guided reductionist in vitro models of neural conversion from hiPSCs. We then discuss early factors relevant in neural patterning, again drawing upon crucial knowledge gained from developmental neurobiological studies. We conclude by discussing open questions relating to these concepts and how their resolution might serve to strengthen the promise of pluripotent stem cells in regenerative medicine.Peer Reviewe

Gene co-expression networks shed light into diseases of brain iron accumulation

Aberrant brain iron deposition is observed in both common and rare neurodegenerative disorders, including those categorized as Neurodegeneration with Brain Iron Accumulation (NBIA), which are characterized by focal iron accumulation in the basal ganglia. Two NBIA genes are directly involved in iron metabolism, but whether other NBIA-related genes also regulate iron homeostasis in the human brain, and whether aberrant iron deposition contributes to neurodegenerative processes remains largely unknown. This study aims to expand our understanding of these iron overload diseases and identify relationships between known NBIA genes and their main interacting partners by using a systems biology approach. We used whole-transcriptome gene expression data from human brain samples originating from 101 neuropathologically normal individuals (10 brain regions) to generate weighted gene co-expression networks and cluster the 10 known NBIA genes in an unsupervised manner. We investigated NBIA-enriched networks for relevant cell types and pathways, and whether they are disrupted by iron loading in NBIA diseased tissue and in an in vivo mouse model. We identified two basal ganglia gene co-expression modules significantly enriched for NBIA genes, which resemble neuronal and oligodendrocytic signatures. These NBIA gene networks are enriched for iron-related genes, and implicate synapse and lipid metabolism related pathways. Our data also indicates that these networks are disrupted by excessive brain iron loading. We identified multiple cell types in the origin of NBIA disorders. We also found unforeseen links between NBIA networks and iron-related processes, and demonstrate convergent pathways connecting NBIAs and phenotypically overlapping diseases. Our results are of further relevance for these diseases by providing candidates for new causative genes and possible points for therapeutic intervention

Genetic and phenotypic characterization of complex hereditary spastic paraplegia

The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease- causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15. Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson’s disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes

iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease

Mutations in PANK2 lead to neurodegeneration with brain iron accumulation. PANK2 has a role in the biosynthesis of coenzyme A (CoA) from dietary vitamin B5, but the neuropathological mechanism and reasons for iron accumulation remain unknown. In this study, atypical patient-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into cortical neuronal cells for studying disease mechanisms in human neurons. We observed no changes in PANK2 expression between control and patient cells, but a reduction in protein levels was apparent in patient cells. CoA homeostasis and cellular iron handling were normal, mitochondrial function was affected; displaying activated NADH-related and inhibited FADH-related respiration, resulting in increased mitochondrial membrane potential. This led to increased reactive oxygen species generation and lipid peroxidation in patient-derived neurons. These data suggest that mitochondrial deficiency is an early feature of the disease process and can be explained by altered NADH/FADH substrate supply to oxidative phosphorylation. Intriguingly, iron chelation appeared to exacerbate the mitochondrial phenotype in both control and patient neuronal cells. This raises caution for the use iron chelation therapy in general when iron accumulation is absent

Mutations in the histone methyltransferase gene KMT2B cause complex early-onset dystonia.

Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.Funding for the project was provided by the Wellcome Trust for UK10K (WT091310) and DDD Study. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund [grant number HICF-1009-003] - see www.ddduk.org/access.html for full acknowledgement. This work was supported in part by the Intramural Research Program of the National Human Genome Research Institute and the Common Fund, NIH Office of the Director. This work was supported in part by the German Ministry of Research and Education (grant nos. 01GS08160 and 01GS08167; German Mental Retardation Network) as part of the National Genome Research Network to A.R. and D.W. and by the Deutsche Forschungsgemeinschaft (AB393/2-2) to A.R. Brain expression data was provided by the UK Human Brain Expression Consortium (UKBEC), which comprises John A. Hardy, Mina Ryten, Michael Weale, Daniah Trabzuni, Adaikalavan Ramasamy, Colin Smith and Robert Walker, affiliated with UCL Institute of Neurology (J.H., M.R., D.T.), King’s College London (M.R., M.W., A.R.) and the University of Edinburgh (C.S., R.W.)

Zerebrale Verarbeitung emotionaler Sprachmelodie - Einfluss akustischer Parameter, des Arousals und die Rolle geschlechtsspezifischer Interaktionen

Nonverbal signals play an important role in the way humans communicate with each other. Body movements like gestures and facial expressions are only one part of it – another important factor is prosody, in the clinical context firstly defined by Monrad-Kohn (1947) as that special facility of language which creates independently from semantics different meanings via modulation of speech-rhythm, loudness, frequency and stress patterns. Approximately, only seven percent of the information about the emotional state of a speaker are inferred from semantics, meaning the content of his words or “what” he or she says. 55 percent is conveyed by body language and the rest, impressive 38 percent, is transported via prosody, e. g. “how” one says, what he says (Mehrabian, 1972). Therefore, prosody – and its adequate interpretation – represents a vital tool within human every-day-life. So far, a lot of research has been carried out to further disentangle the contribution of different acoustic parameters to the expression of emotional prosody. Numerous scientists tried to clarify the influence and importance of single acoustic features within the creation of different emotional intonations (like for example anger, happiness, disgust, sadness and fear). To make the point one can retain an important role for the following acoustic cues in conveying emotional prosody: a) fundamental frequency, b) intensity (also perceived as loudness) and c) temporal information like rhythm and pauses. The following study creates a link between these mainly neuropsychological studies pointing out the importance of different acoustic features in perception and expression of emotional prosody and brain imaging data detecting the neural correlates of processing emotional prosody. Therefore, a neuroimaging experiment was designed which again targeted the voice-processing areas that were previously described as important for elaboration of emotional prosody (Grandjean et al., 2005, Ethofer et al., 2006a), but allowed at the same time to investigate the impact of basic acoustic parameters in the prosodic signal and other possible influences on these areas. Several pre-studies were conducted in order to ensure that the intended emotion within the prosodic signal was reliably recognized (categorization rating) and to assess the valence and arousal of the different stimuli. To obtain an experimental design that is balanced for the valence of the emotional information expressed by prosody, two negative (i.e. anger and fear) and two positive emotions (i.e. happiness and eroticism) were chosen in addition to neutral prosody, serving as control. This design enabled us to investigate a broad spectrum of emotional categories expressed by prosody and their impact on voice-sensitive areas. In the first part of this thesis, differential contributions of certain acoustic parameters on increased responsiveness in right mid STG to emotional versus neutral prosody are investigated (Wiethoff et al., 2008). In this approach separate simple regression analyses of different acoustic parameters were used to partial out their effect on brain activation in voice processing regions. In addition, a multiple regression analysis with all five investigated acoustic parameters was conducted to evaluate the conjoint effect of these parameters on the hemodynamic responses in our target brain areas. The main focus of the second part of the thesis was the investigation of effects that cannot be solely explained by differences in acoustic parameters, but depend on the behavioural relevance of the expressed emotional information (Ethofer et al., 2007). For this aim, the inclusion of stimuli spoken in an erotic tone of voice was of particular importance, since in heterosexual individuals seductively spoken speech-signals gain a high behavioural relevance (e.g. reproduction) as well as an extended meaning when different sexes talk and listen to each other. According to our hypothesis, this fact should enable us to detect behavioural as well as neurophysiological reactions that cannot be exclusively explained on the basis of different acoustic cues, as the physically identical stimuli (with the same variability of the acoustic parameters in it) do or do not gain additional information depending on the gender of speaker and listener. In order to investigate these two aspects the following study was conducted, which is in more detail described in the two publications that form the main part of the manuscript. A general summary that concludes the important findings of both papers will complete this doctoral thesis and can be additionally found in a German version. Since the quotations of the two publications were redundant in some cases, one common list of references is given at the end of the manuscript.Die vorliegende Kernspintomografie-Studie zu emotionaler Prosodie wurde zum besseren Verständnis des Einflusses fünf verschiedener akustischer Parameter auf bestimmte stimmsensitive Areale innerhalb des menschlichen auditorischen Kortex durchgeführt. Darüber hinaus sollte sie klären, ob akustische Parameter alleine für die stärkere Antwort stimmsensitiver Gehirnareale auf emotionale Prosodie (verglichen mit neutraler Sprachmelodie) verantwortlich sind oder ob es über ihren Einfluss hinaus mögliche Wirkungen verhaltensrelevanter Informationen gibt. Als eine der wenigen Studien zur zerebralen Verarbeitung emotionaler Prosodie, die ohne aktive Aufgabe der Probanden durchgeführt wurde, kann sie frühere Befunde zu den an ihr beteiligten Arealen sinnvoll ergänzen und bestätigen (e.g. Wildgruber et al., 2002, 2004 und 2005; Kotz et al., 2003; Mitchell et al., 2003). Darüber hinaus erweiterte die Studie das Spektrum untersuchter emotionaler Intonationen, für die eine erhöhte Antwort stimmsensitiver Areale bereits nachgewiesen war, um die Kategorien “Erotik” und “Angst” und lässt so auf eine generalisierte Rolle des STG bei der Verarbeitung emotionaler Prosodie schliessen. Innerhalb des rechten mittleren STG konnte das vorliegende Experiment den Einfluss der einzelnen akustischen Parameter auf die erhöhte hämodynamische Antwort während der Perzeption emotionaler Prosodie klären. Separate Regressionsanalysen zeigten, dass kein einzelner Parameter die erhöhten Antworten ausreichend erklären konnte, das Zusammenspiel aller fünf analysierten Parameter jedoch sehr wohl. Die gleiche Beobachtung trifft auf das von den Probanden in einer Vorstudie eingeschätzte emotionale Arousal zu: Auch dieser Regressor konnte den Effekt verstärkter hämodynamischer Antworten unserer Zielregion während emotionaler Reize voraussagen. Diese Resultate bestätigen und erweitern unser Verständnis des rechten mittleren STG als eine stimmsensitive Region, die vor allem für das Zusammenspiel mehrerer akustischer Parameter und weniger für einen speziellen Parameter sensibel ist. Plausibel ist in diesem Zusammenhang auch, dass sowohl alle fünf Parameter zusammen, wie auch das zuvor subjektiv eingeschätzte Arousal – welches letztlich ja auch durch die Modulation des akustischen Signals transportiert wird - höhere Aktivierungen während emotionalen Stimuli im rechten mittleren STG erklären konnten. Im ersten Teil der Doktorarbeit ging es um die oben beschriebene Untersuchung dieser stimulusgetriebenen Effekte. Der zweite Teil sollte klären, ob der mittlere STG darüber hinaus ebenfalls für die Dekodierung von Informationen verantwortlich ist, die ihre Bedeutung für den Zuhörer durch die Geschlechterkonstellation der Interaktionspartner erhält. Zur Untersuchung dieser Fragestellung stellen erotisch eingesprochene Reize ein exzellentes Mittel dar, da sie innerhalb der Kommunikation heterosexueller Individuen direkte fortpflanzungsrelevante Informationen tragen können. Der physikalisch-identische Stimulus mit erotischer Intention kann abhängig vom Geschlecht des Sprechers und Zuhörers einen völlig anderen Bedeutungszuwachs bekommen (z. B. ein Signal eines möglichen Partners oder eine weitaus weniger aufregende Äußerung eines Gleichgeschlechtlichen). Erotisch-eingesprochene Reize können somit ausgezeichnet dazu verwendet werden, über physikalische Parameter hinausgehende Veränderungen und deren Auswirkungen auf Verhalten und Gehirnantwort Aufschluss zu geben. Um diese Auswirkungen genauer zu betrachten, wurden sowohl behaviorale Arousalratings als auch hämodynamische Signale des rechten mittleren STG auf erotisch eingesprochene Stimuli gleichgeschlechtlicher Sprecher mit Situationen verglichen, in denen Sprecher und Zuhörer unterschiedlichen Geschlechts waren. Interessanterweise zeigten die Resultate nicht nur höhere Arousalratings auf Verhaltensebene bei der Perzeption erotischer Prosodie, sondern ebenfalls erhöhte hämodynamische Antworten im mittleren rechten STG, wenn die erotisch gesprochenenen Stimuli von einem gegengeschlechtlichen Sprecher stammten. Dieser Effekt zeigte sich exklusiv für erotische Prosodie und konnte für keine der anderen emotionalen Kategorien nachgewiesen werden. Die beiden in dieser Doktorarbeit verwendeten Ansätze konnten also zu einem besseren Verständnis des rechten mittleren STG in sozialer Kommunikation beitragen: Der rechte mittlere superiore temporale Gyrus scheint nicht nur sensitiv für die Modulation prosodisch relevanter akustischer Parameter, sondern ebenfalls dazu befähigt, darüber hinaus gehende Informationen hoher biologischer Relevanz zu dekodieren