Neuronal Differentiation of Induced Pluripotent Stem Cells from Schizophrenia Patients in Two-Dimensional and in Three-Dimensional Cultures Reveals Increased Expression of the Kv4.2 Subunit DPP6 That Contributes to Decreased Neuronal Activity.

Although the molecular underpinnings of schizophrenia (SZ) are still incompletely understood, deficits in synaptic activity and neuronal connectivity have been identified as core pathomechanisms of SZ and other neuropsychiatric disorders. In this study, we generated induced pluripotent stem cell (iPSC) lines from skin fibroblasts from healthy donors and patients diagnosed with idiopathic SZ. We differentiated the human iPSC into cortical neurons both as adherent monolayers and as three-dimensional spheroids. RNA sequencing revealed little overlap in differentially expressed genes between 2D and 3D neuron cultures from SZ iPSC compared with controls. Notably, mRNA transcripts encoding dipeptidyl peptidase-like protein 6 (DPP6), an accessory subunit of Kv4.2 voltage-gated potassium channels, were massively increased in cortical neurons from SZ iPSC in the 2D and 3D model. Consistently, multielectrode array recordings and calcium imaging showed significantly decreased neuronal activity both in 2D and in 3D cultures from SZ neurons. To show a causal relationship, we treated iPSC-derived neurons in 2D cultures with lentiviral DPP6 shRNA vectors and the Kv4.2 channel blocker AmmTx3, respectively. Both treatments successfully reversed neuronal hypoexcitability and hypoactivity in cortical neurons from SZ iPSC. Our data highlight a contribution of DPP6 and Kv4.2 to the deficit in neurotransmission in an iPSC model for SZ, which may be of therapeutic relevance for a subset of SZ patients.

The putative C-type lectin Schlaff ensures epidermal barrier compactness in Drosophila.

The stability of extracellular matrices is in general ensured by cross-linking of its components. Previously, we had shown that the integrity of the layered Drosophila cuticle relies on the presence of a covalent cuticular dityrosine network. Production and composition of this structure remained unstudied. In this work, we present our analyses of the schlaff (slf) gene coding for a putative C-type lectin that is needed for the adhesion between the horizontal cuticle layers. The Slf protein mainly localizes between the two layers called epicuticle and procuticle that separate from each other when the function of Slf is reduced or eliminated paralleling the phenotype of a cuticle with reduced extracellular dityrosine. Localisation of the dityrosinylated protein Resilin to the epicuticle-procuticle interface suggests that the dityrosine network mediates the adhesion of the epicuticle to the procuticle. Ultimately, compromised Slf function is associated with massive water loss. In summary, we propose that Slf is implied in the stabilisation of a dityrosine layer especially between the epicuticle and the procuticle that in turn constitutes an outward barrier against uncontrolled water flow.

Leucine-Rich Repeat Kinase 2 Influences Fate Decision of Human Monocytes Differentiated from Induced Pluripotent Stem Cells.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are strongly associated with familial Parkinson's disease (PD). High expression levels in immune cells suggest a role of LRRK2 in regulating the immune system. In this study, we investigated the effect of the LRRK2 (G2019S) mutation in monocytes, using a human stem cell-derived model expressing LRRK2 at endogenous levels. We discovered alterations in the differentiation pattern of LRRK2 mutant, compared to non-mutant isogenic controls, leading to accelerated monocyte production and a reduction in the non-classical CD14+CD16+ monocyte subpopulation in the LRRK2 mutant cells. LPS-treatment of the iPSC-derived monocytes significantly increased the release of pro-inflammatory cytokines, demonstrating a functional response without revealing any significant differences between the genotypes. Assessment of the migrational capacity of the differentiated monocytes revealed moderate deficits in LRRK2 mutant cells, compared to their respective controls. Our findings indicate a pivotal role of LRRK2 in hematopoietic fate decision, endorsing the involvement of the immune system in the development of PD.

Age-dependent defects of alpha-synuclein oligomer uptake in microglia and monocytes.

Extracellular alpha-synuclein (αsyn) oligomers, associated to exosomes or free, play an important role in the pathogenesis of Parkinson's disease (PD). Increasing evidence suggests that these extracellular moieties activate microglia leading to enhanced neuronal damage. Despite extensive efforts on studying neuroinflammation in PD, little is known about the impact of age on microglial activation and phagocytosis, especially of extracellular αsyn oligomers. Here, we show that microglia isolated from adult mice, in contrast to microglia from young mice, display phagocytosis deficits of free and exosome-associated αsyn oligomers combined with enhanced TNFα secretion. In addition, we describe a dysregulation of monocyte subpopulations with age in mice and humans. Accordingly, human monocytes from elderly donors also show reduced phagocytic activity of extracellular αsyn. These findings suggest that these age-related alterations may contribute to an increased susceptibility to pathogens or abnormally folded proteins with age in neurodegenerative diseases.

6-Hydroxydopamine leads to T2 hyperintensity, decreased claudin-3 immunoreactivity and altered aquaporin 4 expression in the striatum.

The neurotoxin 6-hydroxydopamine (6-OHDA) is frequently used in animal models to mimic Parkinson's disease. Imaging studies describe hyperintense signalling in regions close to the site of the 6-OHDA injection in T2-weighted (T2w) magnetic resonance imaging (MRI). The nature of this hyperintense signal remains elusive and still is matter of discussion. Here we demonstrate hyperintense signalling in T2w MRI and decreased apparent diffusion coefficient (ADC) values following intraventricular injection of 6-OHDA. Moreover, we show decreased GFAP immunoreactivity in brain regions corresponding to the region revealing the hyperintense signalling, probably indicating a loss of astrocytes due to a toxic effect of 6-OHDA. In the striatum, where no hyperintense signalling in MRI was observed following intraventricular 6-OHDA injection, immunohistochemical and molecular analyses revealed an altered expression of the water channel aquaporin 4 and the emergence of vasogenic edema, indicated by an increased perivascular space. Moreover, a significant decrease of claudin-3 immunoreactivity was observed, implying alterations in the blood brain barrier. These findings indicate that intraventricular injection of 6-OHDA results (1) in effects close to the ventricles that can be detected as hyperintense signalling in T2w MRI accompanied by reduced ADC values and (2) in effects on brain regions not adjacent to the ventricles, where a disturbance of water homeostasis occurs. We clearly demonstrate that 6-OHDA leads to brain edema that in turn may affect the overall results of experiments (e.g. behavioral alterations). Therefore, when using 6-OHDA in Parkinson's models effects that are not mediated by degeneration of catecholaminergic neurons have to be considered.