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Development Of A Human iPSC-Derived Cortical Neuron Model Of Adaptor- Protein-Complex-4-Deficiency
(2024)
Adaptor-protein-4-deficiency (AP-4-deficiency) is an autosomal-recessive childhood- onset form of complicated hereditary spastic paraplegia (HSP) caused by bi-allelic loss- of-function mutations in one of the four subunits of the AP-4-complex. These four conditions are named SPG47 (AP4B1, OMIM #614066), SPG50 (AP4M1, OMIM #612936), SPG51 (AP4E1, OMIM #613744) and SPG52 (AP4S1, OMIM #614067), respectively and all present with global developmental delay, progressive spasticity and seizures. Imaging features include a thinning of the corpus callosum, ventriculomegaly and white matter changes. AP-4 is a highly conserved heterotetrameric complex, which is responsible for polarized sorting of transmembrane cargo including the autophagy- related protein 9 A (ATG9A). Loss of any of the four subunits leads to an instable complex and defective sorting of AP-4-cargo. ATG9A is implicated in autophagosome formation and neurite outgrowth. It is missorted in AP-4-deficient cells and CNS-specific knockout of Atg9a in mice results in a phenotype reminiscent of AP-4-deficiency. However, the AP-4-related cellular phenotypes including ATG9A missorting have not been investigated in human neurons.
Thus, the aim of this study is to provide the first human induced pluripotent stem cell- derived (iPSC) cortical neuron model of AP-4-deficiency to explore AP-4-related phenotypes in preparation for a high-content screening. Under the hypothesis that AP-4- deficiency leads to ATG9A missorting, elevated ATG9A levels, impaired autophagy and neurite outgrowth in human iPSC-derived cortical neurons, in vitro biochemical and imaging assays including automated high-content imaging and analysis were applied. First, these phenotypes were investigated in fibroblasts from three patients with compound heterozygous mutations in the AP4B1 gene and their sex-matched parental controls. The same cell lines were used to generate iPSCs and differentiate them into human excitatory cortical neurons.
This work shows that ATG9A is accumulating in the trans-Golgi-network in AP-4- deficient human fibroblasts and that ATG9A levels are increased compared to parental controls and wild type cells suggesting a compensatory mechanism. Protein levels of the AP4E1-subunit were used as a surrogate marker for the AP-4-complex and were decreased in AP-4-deficient fibroblasts with co-immunoprecipitation confirming the instability of the complex. Lentiviral re-expression of the AP4B1-subunit rescues this corroborating the fact that a stable AP-4-complex is needed for ATG9A trafficking. Surprisingly, autophagic flux was present in AP-4-deficient fibroblasts under nutrient- rich and starvation conditions. These phenotypic markers were evaluated in iPSC-derived cortical neurons and here, a robust accumulation of ATG9A in the juxtanuclear area was seen together with elevated ATG9A protein levels. Strikingly, assessment of autophagy markers under nutrient-rich conditions showed alterations in AP-4-deficient iPSC- derived cortical neurons indicating dysfunctional autophagosome formation. These findings point towards a neuron-specific impairment of autophagy and need further investigation. Adding to the range of AP-4-related phenotypes, neurite outgrowth and branching are impaired in AP-4-deficient iPSC-derived cortical neurons as early as 24h after plating and together with recent studies point towards a distinct role of ATG9A in neurodevelopment independent of autophagy.
Together, this work provides the first patient-derived neuron model of AP-4-deficiency and shows that ATG9A is sorted in an AP-4-dependent manner. It establishes ATG9A- related phenotypes and impaired neurite outgrowth as robust markers for a high-content screening. This disease model holds the promise of providing a platform to further study AP-4-deficiency and to search for novel therapeutic targets.
In dieser Dissertation untersuchten wir die neuronalen Korrelate des Training-Effektes einer auditorischen P300 Gehirn-Computer Schnittstelle mittels fMRI Analyse in einem prä-post Design mit zehn gesunden Testpersonen. Wir wiesen in drei Trainings-sitzungen einen Trainingseffekt in der EEG-Analyse der P300 Welle nach und fanden entsprechende Kontraste in einer prä-post Analyse von fMRI Daten, wobei in allen fünf Sitzungen das gleiche Paradigma verwendet wurde. In der fMRI Analyse fanden wir fol-gende Ergebnisse: in einem Target-/ Nichttarget Kontrast zeigte sich verstärkte Aktivie-rung in Generatorregionen der P300 Welle (temporale und inferiore frontale Regionen) und interessanterweise auch in motorassoziierten Arealen, was höhere kognitiver Pro-zesse wie Aufmerksamkeitslenkung und Arbeitsspeicher widerspiegeln könnte. Der Kon-trast des Trainingseffektes zeigte nach dem Training einen stärkeren Rebound Effekt im Sinne einer verstärkten Aktivierung in Generatorregionen der P300 Welle, was eine ver-besserte Erkennung und Prozessierung von Target-Stimuli reflektieren könnte. Eine Ab-nahme von Aktivierung in frontalen Arealen in diesem Kontrast könnte durch effizientere Abläufe kognitiver Prozesse und des Arbeitsgedächtnis erklärt werden.