Katja Nieweg

Marked differences in cholesterol synthesis between neurons and glial cells from postnatal rats.

Neurons have a high demand for cholesterol to develop and maintain membrane‐rich structures like axons, dendrites and synapses, but it remains unclear, whether they can satisfy their need by costly de novo synthesis. To address this, we compared cholesterol synthesis in serum‐free cultures of highly purified CNS neurons and glial cells from postnatal rats. We observed marked cell‐specific differences: Compared with glial cells, neurons showed different profiles of biosynthetic enzymes, post‐squalene precursors and cholesterol metabolites, and they produced cholesterol less efficiently, possibly because of very low levels of lanosterol‐converting enzymes. Astrocytes responded to inhibition of cholesterol synthesis with a much stronger up‐regulation of biosynthetic enzymes than neurons. Our results support the idea that neurons cannot produce cholesterol efficiently and that they depend on an external source of this lipid.

Glia-induced neuronal differentiation by transcriptional regulation

There is increasing evidence that different phases of brain development depend on neuron–glia interactions including postnatal key events like synaptogenesis. To address how glial cells influence synapse development, we analyzed whether and how glia‐derived factors affect gene expression in primary cultures of immunoisolated rat retinal ganglion cells (RGCs) by oligonucleotide microarrays. Our results show that the transcript pattern matched the developmental stage and characteristic properties of RGCs in vitro. Glia‐conditioned medium (GCM) and cholesterol up‐ and downregulated a limited number of genes that influence the development of dendrites and synapses and regulate cholesterol and fatty acid metabolism. The oligonucleotide microarrays detected the transcriptional regulation of neuronal cholesterol homeostasis in response to GCM and cholesterol treatment. Surprisingly, our study revealed neuronal expression and glial regulation of matrix gla protein (Mgp). Together, our results suggest that glial cells promote different aspects of neuronal differentiation by regulating transcription of distinct classes of genes.

Alzheimer's disease-related amyloid- β induces synaptotoxicity in human iPS cell-derived neurons

Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimers disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.

C-terminal fragment of N-cadherin accelerates synapse destabilization by amyloid-β

The aetiology of Alzheimers disease is thought to include functional impairment of synapses and synapse loss as crucial pathological events leading to cognitive dysfunction and memory loss. Oligomeric amyloid-β peptides are well known to induce functional damage, destabilization and loss of brain synapses. However, the complex molecular mechanisms of amyloid-β action resulting ultimately in synapse elimination are incompletely understood, thus limiting knowledge of potential therapeutic targets. Under physiological conditions, long-term synapse stability is mediated by trans-synaptically interacting adhesion molecules such as the homophilically binding N-cadherin/catenin complexes. In this study, we addressed whether inhibition of N-cadherin function affects amyloid-β-induced synapse impairment. We found that blocking N-cadherin function, both by specific peptides interfering with homophilic binding and by expression of a dominant-negative, ectodomain-deleted N-cadherin mutant, resulted in a strong acceleration of the effect of amyloid-β on synapse function in cultured cortical neurons. The frequency of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor-mediated miniature excitatory postsynaptic currents was reduced upon amyloid-β application much earlier than observed in controls. We further hypothesized that ectodomain-shed, transmembrane C-terminal fragments that are generated during N-cadherin proteolytic processing might similarly enhance amyloid-β-induced synapse damage. Indeed, expression of human N-cadherin C-terminal fragment 1 strongly accelerated amyloid-β-triggered synapse impairment. Ectodomain-shed N-cadherin C-terminal fragment 1 is further proteolytically cleaved by γ-secretase. Therefore, both pharmacological inhibition of γ-secretase and expression of the dominant-negative presenilin 1 mutant L166P were used to increase the presence of endogeneous N-cadherin C-terminal fragment 1. Under these conditions, we again found a strong acceleration of amyloid-β-induced synapse impairment, which could be compensated by over-expression of full-length N-cadherin. Intriguingly, western blot analysis of post-mortem brains from patients with Alzheimers disease revealed an enhanced presence of N-cadherin C-terminal fragment 1. Thus, an inhibition of N-cadherin function by proteolytically generated N-cadherin C-terminal fragment 1 might play an important role in Alzheimers disease progression by accelerating amyloid-β-triggered synapse damage.

Asymmetric N-Cadherin Expression Results in Synapse Dysfunction, Synapse Elimination, and Axon Retraction in Cultured Mouse Neurons

Synapse elimination and pruning of axon collaterals are crucial developmental events in the refinement of neuronal circuits. While a control of synapse formation by adhesion molecules is well established, the involvement of adhesion molecules in developmental synapse loss is poorly characterized. To investigate the consequences of mis-match expression of a homophilic synaptic adhesion molecule, we analysed an asymmetric, exclusively postsynaptic expression of N-cadherin. This was induced by transfecting individual neurons in cultures of N-cadherin knockout mouse neurons with a N-cadherin expression vector. 2 days after transfection, patch-clamp analysis of AMPA receptor-mediated miniature postsynaptic currents revealed an impaired synaptic function without a reduction in the number of presynaptic vesicle clusters. Long-term asymmetric expression of N-cadherin for 8 days subsequently led to synapse elimination as indicated by a loss of colocalization of presynaptic vesicles and postsynaptic PSD95 protein. We further studied long-term asymmetric N-cadherin expression by conditional, Cre-induced knockout of N-cadherin in individual neurons in cultures of N-cadherin expressing cortical mouse neurons. This resulted in a strong retraction of axonal processes in individual neurons that lacked N-cadherin protein. Moreover, an in vivo asymmetric expression of N-cadherin in the developmentally transient cortico-tectal projection was indicated by in-situ hybridization with layer V neurons lacking N-cadherin expression. Thus, mis-match expression of N-cadherin might contribute to selective synaptic connectivity.

Embryonic stem cells stably expressing BDNF–GFP exhibit a BDNF-release-dependent enhancement of neuronal differentiation

Summary Brain-derived neurotrophic factor (BDNF) is known to be a crucial regulator of neuronal survival and synaptic plasticity in the mammalian brain. Furthermore, BDNF positively influences differentiation of embryonic neural precursors, as well as that of neural stem cells from adult neurogenic niches. To study the impact of cell-released BDNF on neural differentiation of embryonic stem cells (ESCs), which represent an attractive source for cell transplantation studies, we have generated mouse ESC clones overexpressing BDNF–GFP by use of knock-in technology. After neural differentiation in vitro, we observed that ESC clones overexpressing BDNF–GFP gave rise to an increased number of neurons as compared to control ESCs. Neurons derived from BDNF–GFP-expressing ESCs harbored a more complex dendritic morphology and differentiated into the GABAergic lineage more than controls. Moreover, we show that ESC-derived neurons released BDNF–GFP in an activity-dependent manner and displayed similar electrophysiological properties as cortical neurons. Thus, our study describes the generation of ESCs stably overexpressing BDNF–GFP, which are ideally suited to investigate the ameliorating effects of BDNF in cell transplantation studies of various neuropathological conditions.

mESC-Based in vitro Differentiation Models to Study Vascular Response and Functionality Following Genotoxic Insults

Because of high exposure to systemic noxae, vascular endothelial cells (EC) have to ensure distinct damage defense and regenerative mechanisms to guarantee vascular health. For meaningful toxicological drug assessments employing embryonic stem cell (ESC)-based in vitro models, functional competence of differentiated progeny and detailed knowledge regarding damage defense mechanisms are essential. Here, mouse ESCs (mESC) were differentiated into functionally competent vascular cells (EC and smooth muscle cells [SMC]). mESC, EC, and SMC were comparatively analyzed regarding DNA repair and DNA damage response (DDR). Differentiation was accompanied by both congruent and unique alterations in repair and DDR characteristics. EC and SMC shared the downregulation of genes involved cell cycle regulation and repair of DNA double-strand breaks (DSBs) and mismatches, whereas genes associated with nucleotide excision repair (NER), apoptosis, and autophagy were upregulated when compared with mESC. Expression of genes involved in base excision repair (BER) was particularly low in SMC. IR-induced formation of DSBs, as detected by nuclear γH2AX foci formation, was most efficient in SMC, the repair of DSBs was fastest in EC. Together with substantial differences in IR-induced phosphorylation of p53, Chk1, and Kap1, the data demonstrate complex alterations in DDR capacity going along with the loss of pluripotency and gain of EC- and SMC-specific functions. Notably, IR exposure of early vascular progenitors did not impair differentiation into functionally competent EC and SMC. Summarizing, mESC-based vascular differentiation models are informative to study the impact of environmental stressors on differentiation and function of vascular cells.

Lab Resource: Stem Cell LineEpisomal plasmid-based generation of an iPSC line from an 83-year-old individual carrying the APOE4/4 genotype: i10984

In this study, lymphoblastoid cells derived from a 83-year old individual with a 15year history of progressive presenile dementia, were used to generate iPS cells, employing episomal plasmids expressing OCT4, SOX2, LIN28, L-MYC and a p53 shRNA. The individual was homozygous for the APOE4 allele. The resulting iPS cells had a normal karyotype, retained the APOE4/4 genotype, expressed pluripotency markers, were free of genomically integrated plasmids, and could be differentiated into cell type representatives from the three germ layers in vitro.

Episomal plasmid-based generation of an iPSC line from a 79-year-old individual carrying the APOE4/4 genotype: i11001.

In this study, lymphoblastoid cells derived from a 79-year old individual with a history of progressive presenile dementia, were used to generate iPS cells, employing episomal plasmids expressing OCT4, SOX2, KLF4, LIN28, L-MYC and a p53 shRNA. The individual was homozygous for the APOE4 allele. The resulting iPS cells had a normal karyotype, retained the APOE4/4 genotype, expressed pluripotency markers, were free of genomically integrated plasmids, and could be differentiated into cell type representatives from the three germ layers in vitro.