Genetical analysis of neuronal glycine transporter GlyT1b function and its transcriptional regulation in vivo
Final Report Abstract
In the central nervous system glycine acts as a neurotransmitter both at inhibitory glycinergic synapses and at NMDA receptor containing glutamatergic synapses. The extracellular glycine concentration is regulated by two high affinity/large capacity transporters the GlyTs. GlyT1 is expressed both in a subpopulation of presumably glutamaterigic neurons as well as in many astrocytes throughout the central nervous system. It has been proposed previously that depending on the cell type, brain region and developmental stage GlyT1 might have different functions and these differences might correlate with different GlyT1 isoforms that are all expressed from a single gene. In this project we have generated a mouse line, that carries a knock-in of a luciferase-RFP fusion protein encoding sequence into the exon 1b of the endogenous GlyT1 gene, thereby creating a functional knock out of the GlyT1 isoforms GlyT1 b and c. Additionally, this knock-in was predicted to function as a reporter mouse-line of these GlyT1 isoforms whereas GlyT1a expression was predicted to be unchanged. GlyT1b/cki/ki mice were born at the expected mendelian ratios and did not display a severe phenotype at birth and reached adulthood without increased mortality. During postnatal development, however, GlyT1b/cki/ki mice gained less weight, appeared generally hypoactive and developed brief episodes of tremor and muscular rigidity followed by extensive periods, demonstrating an up to now unrecognized role of GlyT1 for the function of the mature central nervous system. Brain lysates from GlyT1b/cki/ki mice showed high luciferase activity in brain stem and spinal cord, i.e. regions known to be rich in glycinergic neurotransmission, whereas brain regions known to harbour large neuronal GlyT1 populations showed only low luciferase expression. The level of luciferase expression correlated well with the region specific impact of the GlyT1 KI onto the total GlyT1 activity and protein expression. Interestingly, reduction of GlyT1 protein was more pronounced in adult animals as compared to neonatal animals, suggesting, a developmental upregulation of GlyT1b/c during postnatal development. qPCR analyses however, demonstrated a significant albeit moderate effect of the Glyt1 gene modification on the GlyT1a gene function. Immunohistochemical analysis revealed a general reduction in the expression level of GlyT1, suggesting that the expression of GlyT1b/c is not as assumed previously cell type specific. Measurement of the cerebrospinal fluid (CSF) and serum glycine concentration showed only moderate increase in the CSF glycine concentration similar to that observed in asymptomatic adult glial GlyT1 deficient mice, suggesting regional differences in the extracellular glycine concentration in both mouse strains. Interestingly, similar observation was made in patients suffering from GHlyT1 encephalopathy, which was associated with functionals defects in GlyT1, and causing severe respiratory deficits. Analysis of the in-vivo respiratory activity in GlyT1b/cki/ki mice revealed a reduced amplitude and variability of the breathing rhythm suggesting that GlyT1 is important to maintain the modulatory (glycine dependent) input onto the circuitry important for the generation of the breathing rhythm. Interestingly, electrophysiological recording of glycinergic neurotransmission in the brainstem auditory nuclei revealed only moderate differences between genotype that did not suffice to explain the phenotype seen in GlyT1b/cki/ki mice. Whether these moderate effects of partial GlyT1 deficiency are a result from the specialized synapse architecture in the auditory system tuned for high frequency stimulation, is unclear at present. Taken together our data provide evidence for a previously unrecognized role of GlyT1 in the mature nervous system. Future studies will have to address the precise mechanisms and the precise cell types involved in the ontogenesis of the phenotype seen in GlyT1b/cki/ki mice.
Publications
- (2016) Loss of Glycine Transporter 1 Causes a Subtype of Glycine Encephalopathy with Arthrogryposis and Mildly Elevated Cerebrospinal Fluid Glycine. Am J Hum Genet. 99, 1172-1180
Kurolap, A., Armbruster, A., Hershkovitz, T., Hauf, K., Mory, A., Paperna, T., Hannappel, E., Tal, G., Nijem, Y., Sella, E., Mahajnah, M., Ilivitzki, A., Hershkovitz, D., Ekhilevitch, N., Mandel, H., Eulenburg, V., Baris, H. N.
(See online at https://doi.org/10.1016/j.ajhg.2016.09.004) - (2018) GlyT1 determines the glycinergic phenotype of amacrine cells in the mouse retina. Brain Struct Funct
Eulenburg, V., Knop, G., Sedmak, T., Schuster, S., Hauf, K., Schneider, J., Feigenspan, A., Joachimsthaler, A., Brandstätter, J. H.
(See online at https://doi.org/10.1007/s00429-018-1684-3)
