TY  - JOUR
A1  - Piro, Inken
A1  - Eckes, Anna-Lena
A1  - Kasaragod, Vikram Babu
A1  - Sommer, Claudia
A1  - Harvey, Robert J.
A1  - Schaefer, Natascha
A1  - Villmann, Carmen
T1  - Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease
JF  - Frontiers in Molecular Neuroscience
N2  - Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC\(_{50}\) value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.
KW  - glycine receptor
KW  - hyperekplexia
KW  - startle disease
KW  - gephyrin
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-246676
SN  - 1662-5099
VL  - 14
ER  - 
TY  - JOUR
A1  - Fischhaber, Natalie
A1  - Faber, Jessica
A1  - Bakirci, Ezgi
A1  - Dalton, Paul D.
A1  - Budday, Silvia
A1  - Villmann, Carmen
A1  - Schaefer, Natascha
T1  - Spinal Cord Neuronal Network Formation in a 3D Printed Reinforced Matrix-A Model System to Study Disease Mechanisms
JF  - Advanced Healthcare Materials
N2  - 3D cell cultures allow a better mimicry of the biological and mechanical environment of cells in vivo compared to 2D cultures. However, 3D cell cultures have been challenging for ultrasoft tissues such as the brain. The present study uses a microfiber reinforcement approach combining mouse primary spinal cord neurons in Matrigel with melt electrowritten (MEW) frames. Within these 3D constructs, neuronal network development is followed for 21 days in vitro. To evaluate neuronal development in 3D constructs, the maturation of inhibitory glycinergic synapses is analyzed using protein expression, the complex mechanical properties by assessing nonlinearity, conditioning, and stress relaxation, and calcium imaging as readouts. Following adaptation to the 3D matrix-frame, mature inhibitory synapse formation is faster than in 2D demonstrated by a steep increase in glycine receptor expression between days 3 and 10. The 3D expression pattern of marker proteins at the inhibitory synapse and the mechanical properties resemble the situation in native spinal cord tissue. Moreover, 3D spinal cord neuronal networks exhibit intensive neuronal activity after 14 days in culture. The spinal cord cell culture model using ultrasoft matrix reinforced by MEW fibers provides a promising tool to study and understand biomechanical mechanisms in health and disease.
KW  - 3D cell cultures
KW  - spinal cord neurons
KW  - neuronal networks
KW  - mouse
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256353
VL  - 10
IS  - 19
ER  -