Abstract
Amyloid Precursor Protein (APP) is concentrated at neuronal synapses and can be proteolytically cleaved to release soluble secreted Amyloid Precursor Protein alpha (sAPPa) into the extracellular space. Exogenously applied sAPPa can rescue memory defects, promote neurogenesis, promote neurite outgrowth, and enhance Long Term Potentiation (LTP) in cell culture, brain slices and whole animal studies. The associated genes and proteins linked to these potentiating, neurogenic and neuroprotective mechanisms are largely unknown but sAPPa is being considered as a potential Alzheimer’s Disease therapy and therefore understanding its effect on the molecular biology of neurons and astrocytes will be useful. This study primarily analysed changes in the proteome and transcriptome of neurons and astrocytes after treatment with sAPPa. The secondary aim was to develop a human astrocyte cell culture model, which will be used to study the effects of sAPPa on this supportive neural cell and be available for other researchers use.
Using RNA sequencing (RNAseq), and Sequential Window of All THeoretical Spectra Mass Spectrometry (SWATH-MS), gene transcripts and proteins respectively, were identified and quantified from human cortical neurons, and proteins (proteome and secretome) from mouse primary astrocyte cell cultures. Differentially expressed transcripts and proteins when cells were treated with 1 nM sAPPa over a time course, were identified. The majority of those differentially expressed molecules have biological functions associated with synaptic transmission and a significant number have been highlighted for further investigation.
In the primary cell astrocytes, the differentially expressed proteins more broadly formed groups in functional categories: actin dynamics including neurite/filipodia outgrowth and cytoskeletal morphology, microtubule dynamics, vesicle dynamics (including receptor trafficking, docking and fusion and synaptic transmission), myelination, cell signalling, autophagy, metabolism, transcription, and post translational modification. The aim of producing a human neural cell model for astrocytes was successful as a genetically modified iPSC cell line was generated, which can be induced to differentiate into cells that express astrocyte proteins. Further characterisation of these cells and how sAPPa affects their transcriptome and proteome can now be carried out.
In the human neurons in culture, enriched Gene Ontology (GO) terms in the proteome mirrored what was observed in the differentially expressed transcripts from the transcriptome (and both proteome and secretome astrocyte studies), namely: actin and microtubule dynamics (cytoskeleton), cell-cell and cell-extra cellular matrix (ECM) interaction, neurotransmitter, receptor and ion channel (protein) trafficking, cell signalling, vesicle dynamics, and the proteosome.
These data allow the assembly of a comprehensive portfolio of transcripts and proteins affected by sAPPa (sAPPa-tome) and as a result, the hypothesis that sAPPa causes changes to protein levels in astrocytes and neurons that are involved with neurological functions associated with learning and memory, has been considerably strengthened.