Author Correction: Parvalbumin expression in oligodendrocyte-like CG4 cells causes a reduction in mitochondrial volume, attenuation in reactive oxygen species production and a decrease in cell processes' length and branching.

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

Parvalbumin expression in oligodendrocyte-like CG4 cells causes a reduction in mitochondrial volume, attenuation in reactive oxygen species production and a decrease in cell processes' length and branching.

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.

Correction to: Four novel interaction partners demonstrate diverse modulatory effects on voltage-gated CaV2.2 Ca2+ channels.

The article was originally published with one author missing. The name of the co-author Roman Moravcik was inadvertently omitted. His name and affiliation have now been added to the author list. The original article has been corrected.

Four novel interaction partners demonstrate diverse modulatory effects on voltage-gated CaV2.2 Ca2+ channels.

Voltage-gated Ca2+ channels are embedded in a network of protein interactions that are fundamental for channel function and modulation. Different strategies such as high-resolution quantitative MS analyses and yeast-two hybrid screens have been used to uncover these Ca2+ channel nanodomains. We applied the yeast split-ubiquitin system with its specific advantages to search for interaction partners of the CaV2.2 Ca2+ channel and identified four proteins: reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS) and transmembrane protein 223 (TMEM223). Interactions were verified using the yeast split-ubiquitin system and narrowed down to CaV2.2 domain IV. Colocalization studies using fluorescent constructs demonstrated defined regions of subcellular localization. Detailed electrophysiological studies revealed that coexpression of RTN1 modulated CaV2.2 channels only to a minor extent. SLC38 accelerated the cumulative current inactivation during a high-frequency train of brief depolarizing pulses. As neurons expressing CaV2.2 channels were exposed to high-frequency bursts under physiological conditions, observed regulation may have a negative modulatory effect on transmitter release. Coexpression of PTGDS significantly lowered the average current density and slowed the kinetics of cumulative current inactivation. Since the latter effect was not significant, it may only partly compensate the first one under physiological conditions. Expression of TMEM223 lowered the average current density, accelerated the kinetics of cumulative current inactivation and slowed the kinetics of recovery from inactivation. Therefore, TMEM223 and, to a lesser extent, PTGDS, may negatively modulate Ca2+ entry required for transmitter release and/or for dendritic plasticity under physiological conditions.

Parvalbumin alters mitochondrial dynamics and affects cell morphology.

The Ca2+-binding protein parvalbumin (PV) and mitochondria play important roles in Ca2+ signaling, buffering and sequestration. Antagonistic regulation of PV and mitochondrial volume is observed in in vitro and in vivo model systems. Changes in mitochondrial morphology, mitochondrial volume and dynamics (fusion, fission, mitophagy) resulting from modulation of PV were investigated in MDCK epithelial cells with stable overexpression/downregulation of PV. Increased PV levels resulted in smaller, roundish cells and shorter mitochondria, the latter phenomenon related to reduced fusion rates and decreased expression of genes involved in mitochondrial fusion. PV-overexpressing cells displayed increased mitophagy, a likely cause for the decreased mitochondrial volumes and the smaller overall cell size. Cells showed lower mobility in vitro, paralleled by reduced protrusions. Constitutive PV down-regulation in PV-overexpressing cells reverted mitochondrial morphology and fractional volume to the state present in control MDCK cells, resulting from increased mitochondrial movement and augmented fusion rates. PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial volume.

Compound heterozygous SPATA5 variants in four families and functional studies of SPATA5 deficiency.

Variants in the SPATA5 gene were recently described in a cohort of patients with global developmental delay, sensorineural hearing loss, seizures, cortical visual impairment and microcephaly. SPATA5 protein localizes predominantly in the mitochondria and is proposed to be involved in mitochondrial function and brain developmental processes. However no functional studies have been performed. This study describes five patients with psychomotor developmental delay, microcephaly, epilepsy and hearing impairment, who were thought clinically to have a mitochondrial disease with subsequent whole-exome sequencing analysis detecting compound heterozygous variants in the SPATA5 gene. A summary of clinical data of all the SPATA5 patients reported in the literature confirms the characteristic phenotype. To assess SPATA5's role in mitochondrial dynamics, functional studies were performed on rat cortical neurons. SPATA5-deficient neurons had a significant imbalance in the mitochondrial fusion-fission rate, impaired energy production and short axons. In conclusion, SPATA5 protein has an important role in mitochondrial dynamics and axonal growth. Biallelic variants in the SPATA5 gene can affect mitochondria in cortical neurons and should be considered in patients with a neurodegenerative disorder and/or with clinical presentation resembling a mitochondrial disorder.

Ca(V)1.2 and Ca(V)1.3 L-type calcium channels regulate the resting membrane potential but not the expression of calcium transporters in differentiated PC12 cells.

PC12 cells differentiated under the influence of the neuronal growth factor (NGF) serve as a model of both sympathetic neurons and chromaffin cells. NGF-induced differentiation critically depends on elevated intracellular calcium concentration. Main pathway for Ca²âº entry in excitable cells is represented by voltage-dependent calcium channels including L-type calcium channels (LTCC). We investigated role of Ca(V)1.2 and Ca(V)1.3 LTCC subtypes in NGF-differentiated PC12 cells. The expression of LTCC subtypes was downregulated by transfection of NGF-differentiated PC12 cells with siRNA for either CACNA1C or CACNA1D gene. Efficiency of gene silencing was verified by RT-PCR and by functional essay. The dominant LTCC subtype in PC12 cells was Ca(V)1.2. Downregulation of either LTCC significantly hyperpolarized the resting membrane potential. Expression of mRNA for intracellular calcium transporters inositol trisphosphate receptor type 1, 2 and 3, ryanodine receptor type 1 and 2 and sarco/endoplasmic reticulum Ca²âº ATPase type 2 as well as plasma membrane transporters Na⁺-Ca²âº exchanger type 1 and 2 was not altered in the absence of either LTCC subtype. In conclusion, Ca²âº influx through Ca(V)1.2 or to Ca(V)1.3 channel subtypes contributes to maintenance of the resting membrane potentials of NGF-differentiated PC12 cells but is not required for regulation of expression of genes for calcium-transporting proteins.

Tetraspanin-13 modulates voltage-gated CaV2.2 Ca2+ channels.

Integration of voltage-gated Ca(2+) channels in a network of protein-interactions is a crucial requirement for proper regulation of channel activity. In this study, we took advantage of the specific properties of the yeast split-ubiquitin system to search for and characterize so far unknown interaction partners of CaV2 Ca(2+) channels. We identified tetraspanin-13 (TSPAN-13) as an interaction partner of the α1 subunit of N-type CaV2.2, but not of P/Q-type CaV2.1 or L- and T-type Ca(2+) channels. Interaction could be located between domain IV of CaV2.2 and transmembrane segments S1 and S2 of TSPAN-13. Electrophysiological analysis revealed that TSPAN-13 specifically modulates the efficiency of coupling between voltage sensor activation and pore opening of the channel and accelerates the voltage-dependent activation and inactivation of the Ba(2+) current through CaV2.2. These data indicate that TSPAN-13 might regulate CaV2.2 Ca(2+) channel activity in defined synaptic membrane compartments and thereby influences transmitter release.

NGF-induced neurite outgrowth in PC12 cells is independent of calcium entry through L-type calcium channels.

Neuronal growth factor (NGF) induces neurodifferentiation of PC12 cells into cholinergic neurons-like cells. It was shown that intracellular Ca2+ ions participate in regulation of the differentiation of PC12 cells. We tested whether L-type calcium channels contribute to Ca2+ entry which supports neurite outgrowth accompanying NGF-activated differentiation process. Development of morphological changes did correlate with increase of functional expression of L-type calcium channels. However, inhibition of L-type calcium channels by 1 µM of isradipine did not affect significantly an NGF-activated neurite outgrowth.