Beat Schwaller

Malignant mesothelioma (MM) is a currently incurable, aggressive cancer derived from mesothelial cells, most often resulting from asbestos exposure. The current first-line treatment in unresectable MM is cisplatin/pemetrexed, which shows very little long-term effectiveness, necessitating research for novel therapeutic interventions. The existing chemotherapies often act on the cytoskeleton, including actin filaments and microtubules, but recent advances indicate the ‘fourth’ form consisting of the family of septins, representing a novel target. The septin inhibitor forchlorfenuron (FCF) and FCF analogs inhibit MM cell growth in vitro, but at concentrations which are too high for clinical applications. Based on the reported requirement of the chloride group in the 2-position of the pyridine ring of FCF for MM cell growth inhibition and cytotoxicity, we systematically investigated the importance (cell growth-inhibiting capacity) of the halogen atoms fluorine, chlorine, bromine and iodine in the 2- or 3-position of the pyridine ring. The MM cell lines ZL55, MSTO-211H, and SPC212, and—as a control—immortalized Met-5A mesothelial cells were used. The potency of the various halogen substitutions in FCF was mostly correlated with the atom size (covalent radius); the small fluoride analogs showed the least effect, while the largest one (iodide) most strongly decreased the MTT signals, in particular in MM cells derived from epithelioid MM. In the latter, the strongest effects in vitro were exerted by the 2-iodo and, unexpectedly, the 2-trifluoromethyl (2-CF₃) FCF analogs, which were further tested in vivo in mice. However, FCF-2-I and, more strongly, FCF-2-CF₃ caused rapidly occurring strong symptoms of systemic toxicity at doses lower than those previously obtained with FCF. Thus, we investigated the effectiveness of FCF (and selected analogs) in vitro in MM cells which were first exposed to cisplatin. The slowly appearing population of cisplatin-resistant cells was still susceptible to the growth-inhibiting/cytotoxic effect of FCF and its analogs, indicating that cisplatin and FCF target non-converging pathways in MM cells. Thus, a combination therapy of cisplatin and FCF (analogs) might represent a new avenue for the treatment of repopulating chemo-resistant MM cells in this currently untreatable cancer.

An amendment to this paper has been published and can be accessed via the original article.

The prevalence of autism spectrum disorder (ASD)—a type of neurodevelopmental disorder—is increasing and is around 2% in North America, Asia, and Europe. Besides the known genetic link, environmental, epigenetic, and metabolic factors have been implicated in ASD etiology. Although highly heterogeneous at the behavioral level, ASD comprises a set of core symptoms including impaired communication and social interaction skills as well as stereotyped and repetitive behaviors. This has led to the suggestion that a large part of the ASD phenotype is caused by changes in a few and common set of signaling pathways, the identification of which is a fundamental aim of autism research. Using advanced bioinformatics tools and the abundantly available genetic data, it is possible to classify the large number of ASD-associated genes according to cellular function and pathways. Cellular processes known to be impaired in ASD include gene regulation, synaptic transmission affecting the excitation/inhibition balance, neuronal Ca²⁺ signaling, development of short-/long-range connectivity (circuits and networks), and mitochondrial function. Such alterations often occur during early postnatal neurodevelopment. Among the neurons most affected in ASD as well as in schizophrenia are those expressing the Ca²⁺-binding protein parvalbumin (PV). These mainly inhibitory interneurons present in many different brain regions in humans and rodents are characterized by rapid, non-adaptive firing and have a high energy requirement. PV expression is often reduced at both messenger RNA (mRNA) and protein levels in human ASD brain samples and mouse ASD (and schizophrenia) models. Although the human PVALB gene is not a high-ranking susceptibility/risk gene for either disorder and is currently only listed in the SFARI Gene Archive, we propose and present supporting evidence for the Parvalbumin Hypothesis, which posits that decreased PV level is causally related to the etiology of ASD (and possibly schizophrenia).

In neurodevelopmental disorders (NDD) including autism spectrum disorder (ASD) and schizophrenia, impairment/malfunctioning of a subpopulation of interneurons expressing the calcium-binding protein parvalbumin (PV) –here termed Pvalb neurons– has gradually emerged as a possible cause. These neurons may represent a hub or point-of-convergence in the etiology of NDD. Increased oxidative stress associated with mitochondria impairment in Pvalb neurons is discussed as an essential step in schizophrenia etiology. Since PV downregulation is a common finding in ASD and schizophrenia individuals and PV-deficient (PV-/-) mice show a strong ASD-like behavior phenotype, we investigated the putative link between PV expression, alterations in mitochondria and oxidative stress. In a longitudinal study with 1, 3 and 6-months old PV-/- and wildtype mice, oxidative stress was investigated in 9 Pvalb neuron subpopulations in the hippocampus, striatum, somatosensory cortex, medial prefrontal cortex, thalamic reticular nucleus (TRN) and cerebellum. In Pvalb neuron somata in the striatum and TRN, we additionally determined mitochondria volume and distribution at these 3 time points. In all Pvalb neuron subpopulations, we observed an age-dependent increase in oxidative stress and the increase strongly correlated with PV expression levels, but not with mitochondria density in these Pvalb neurons. Moreover, oxidative stress was elevated in Pvalb neurons of PV-/- mice and the magnitude of the effect was again correlated with PV expression levels in the corresponding wildtype Pvalb neuron subpopulations. The PV-dependent effect was insignificant at 1 month and relative differences between WT and PV-/- Pvalb neurons were largest at 3 months. Besides the increase in mitochondria volume in PV’s absence in TRN and striatal PV-/- Pvalb neurons fully present already at 1 month, we observed a redistribution of mitochondria from the perinuclear region towards the plasma membrane at all time points. We suggest that in absence of PV, slow Ca2+ buffering normally exerted by PV is compensated by a (mal)adaptive, mostly sub-plasmalemmal increase in mitochondria resulting in increased oxidative stress observed in 3- and 6-months old mice. Since PV-/- mice display core ASD-like symptoms already at 1 month, oxidative stress in Pvalb neurons is not a likely cause for their ASD-related behavior observed at this age.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background: In fast firing, parvalbumin (PV)-expressing (Pvalb) interneurons, PV acts as an intracellular Ca²⁺ signal modulator with slow-onset kinetics. In Purkinje cells of PV^−/− mice, adaptive/homeostatic mechanisms lead to an increase in mitochondria, organelles equally capable of delayed Ca²⁺ sequestering/buffering. An inverse regulation of PV and mitochondria likewise operates in cell model systems in vitro including myotubes, epithelial cells, and oligodendrocyte-like cells overexpressing PV. Whether such opposite regulation pertains to all Pvalb neurons is currently unknown. In oligodendrocyte-like cells, PV additionally decreases growth and branching of processes in a cell-autonomous manner. Methods: The in vivo effects of absence of PV were investigated in inhibitory Pvalb neurons expressing EGFP, present in the somatosensory and medial prefrontal cortex, striatum, thalamic reticular nucleus, hippocampal regions DG, CA3, and CA1 and cerebellum of mice either wild-type or knockout (PV^−/−) for the Pvalb gene. Changes in Pvalb neuron morphology and PV concentrations were determined using immunofluorescence, followed by 3D-reconstruction and quantitative image analyses. Results: PV deficiency led to an increase in mitochondria volume and density in the soma; the magnitude of the effect was positively correlated with the estimated PV concentrations in the various Pvalb neuron subpopulations in wild-type neurons. The increase in dendrite length and branching, as well as thickness of proximal dendrites of selected PV^−/− Pvalb neurons is likely the result of the observed increased density and length of mitochondria in these PV^−/− Pvalb neuron dendrites. The increased branching and soma size directly linked to the absence of PV is assumed to contribute to the increased volume of the neocortex present in juvenile PV^−/− mice. The extended dendritic branching is in line with the hypothesis of local hyperconnectivity in autism spectrum disorder (ASD) and ASD mouse models including PV^−/− mice, which display all ASD core symptoms and several comorbidities including cortical macrocephaly at juvenile age. Conclusion: PV is involved in most proposed mechanisms implicated in ASD etiology: alterations in Ca²⁺ signaling affecting E/I balance, changes in mitochondria structure/function, and increased dendritic length and branching, possibly resulting in local hyperconnectivity, all in a likely cell autonomous way.

Introduction: Alzheimer's disease (AD) is linked to neuronal calcium dyshomeostasis, which is associated with network hyperexcitability. Decreased expression of the calcium‐binding protein cal‐ bindin‐D_28K (CB) might be a susceptibility factor for AD. The subiculum is affected early in AD, for unknown reasons.Methods: In AD, CB knock‐out and control mice fluorescence Ca²⁺ imaging combined with patch clamp were used to characterize Ca²⁺ dynamics, resting Ca²⁺, and Ca²⁺‐buffering capacity in subicular neurons. CB expression levels in wild‐type and AD mice were also analyzed.Results: The subiculum and dentate gyrus of wild‐type mice showed age‐related decline in CB expression not observed in AD mice. Resting Ca²⁺ and Ca²⁺‐buffering capacity was increased in aged AD mice subicular dendrites. Modeling suggests that AD calcium changes can be explained by alterations of Ca²⁺ extrusion pumps rather than by buffers.Discussion: Overall, abnormal Ca²⁺ homeostasis in AD has an age dependency that comprises multiple mechanisms, including compensatory processes.

Oncotarget | https://doi.org/10.18632/oncotarget.27341 Walter Blum, Thomas Henzi, László Pecze, Kim-Long Diep, Christian G. Bochet, Beat Schwaller

Malignant mesothelioma (MM) is an aggressive asbestos-linked neoplasm, characterized by dysregulation of signaling pathways. Due to intrinsic or acquired chemoresistance, MM treatment options remain limited. Calretinin is a Ca²⁺-binding protein expressed during MM tumorigenesis that activates the FAK signaling pathway, promoting invasion and epithelial-to-mesenchymal transition. Constitutive calretinin downregulation decreases MM cells’ growth and survival, and impairs tumor formation in vivo. In order to evaluate early molecular events occurring during calretinin downregulation, we generated a tightly controlled IPTG-inducible expression system to modulate calretinin levels in vitro. Calretinin downregulation significantly reduced viability and proliferation of MM cells, attenuated FAK signaling and reduced the invasive phenotype of surviving cells. Importantly, surviving cells showed a higher resistance to cisplatin due to increased Wnt signaling. This resistance was abrogated by the Wnt signaling pathway inhibitor 3289-8625. In various MM cell lines and regardless of calretinin expression levels, blocking of FAK signaling activated the Wnt signaling pathway and vice versa. Thus, blocking both pathways had the strongest impact on MM cell proliferation and survival. Chemoresistance mechanisms in MM cells have resulted in a failure of single-agent therapies. Targeting of multiple components of key signaling pathways, including Wnt signaling, might be the future method-of-choice to treat MM.

Forebrain glial cells - ependymal cells and astrocytes -acquire upon injury- a “reactive” phenotype associated with parvalbumin (PV) upregulation. Since free radicals, e.g. reactive oxygen species (ROS) play a role in the pathogenesis of multiple sclerosis, and that PV-upregulation in glial cells is inversely correlated with the level of oxidative stress, we hypothesized that PV-upregulation might also protect oligodendrocytes by decreasing ROS production. Lentiviral transduction techniques allowed for PV overexpression in CG4 oligodendrocyte progenitor cells (OPCs). Depending on the growth medium CG4 cells can be maintained in an OPC-like state, or induced to differentiate into an oligodendrocyte (OLG)-like phenotype. While increased levels of PV had no effect on cell proliferation and invasiveness in vitro, PV decreased the mitochondria volume in CG4 cell bodies, as well as the mitochondrial density in CG4 processes in both OPC-like and OLG-like states. In line with the PV-induced global decrease in mitochondrial volume, elevated PV levels reduced transcript levels of mitochondrial transcription factors involved in mitochondria biogenesis. In differentiated PV-overexpressing CG4 cells with a decreased mitochondrial volume, UV-induced ROS production was lower than in control CG4 cells hinting towards a possible role of PV in counteracting oxidative stress. Unexpectedly, PV also decreased the length of processes in undifferentiated CG4 cells and moreover diminished branching of differentiated CG4 cell processes, strongly correlated with the decreased density of mitochondria in CG4 cell processes. Thus besides conferring a protective role against oxidative stress, PV in a cell autonomous fashion additionally affects process’ growth and branching in CG4 cells.