Brain Microvasculature Defects and Glut1 Deficiency Syndrome Averted by Early Repletion of the Glucose Transporter-1 Protein
Mutations in the SLC2A1 gene evolve into the rare but often incapacitating pediatric neurodevelopmental disorder, Glut1 deficiency syndrome (Glut1 DS)1,2. Initially considered exceptionally rare, reports that SLC2A1 mutations account for ∼1% of idiopathic generalized epilepsies and the recognition of an expanding Glut1 DS phenotype suggest that there may be in excess of 11,000 individuals afflicted with the disorder in the US alone3,4. Patients with classic Glut1 DS suffer low brain glucose levels and exhibit a phenotype characterized by early-onset seizures, delayed development, acquired microcephaly (decelerating head growth) and a complex movement disorder combining features of spasticity, ataxia and dystonia5,6. Low concentration of glucose in the cerebrospinal fluid (CSF), also known as hypoglycorrhachia, is the most reliable biomarker of the disease2. The disease characteristics of Glut1 DS are consistent with the widespread but especially abundant expression of Glut1 in the endothelial cells (ECs) of the brain microvasculature7, where the protein facilitates the transport of blood glucose across the blood–brain barrier (BBB) to the CNS.
Although the genetic cause of Glut1 DS was identified almost two decades ago and notwithstanding widespread interest in Glut1 biology, little is known about the precise molecular and cellular pathology underlying the human disorder. Nor is there an optimal treatment for Glut1 DS. Clinicians have so far relied mostly on the ketogenic diet8,9. However, the diet is, at best, partially effective, mitigating seizure activity in some young patients but unable to attenuate virtually any other neurological deficit10.
We modelled Glut1 DS in mice by inactivating one copy of the murine Slc2a1 gene11. Mutants display many of the signature features of human Glut1 DS including seizure activity, hypoglycorrhachia, micrencephaly and impaired motor performance. Here we link overt manifestations of brain dysfunction in the mutants to profound defects of the cerebral microvasculature. We demonstrate that low Glut1 protein not only delays brain angiogenesis but also triggers microvasculature diminution, without affecting BBB integrity. Repletion of the protein in neonatal Glut1 DS model mice ensures the proper development of the brain microvasculature and preserves it during adulthood. Moreover, seizures and disease progression in these mice is rapidly arrested. Restoring the protein to 2-week old mutants, in which certain disease characteristics are readily apparent, is less effective in shaping normal brain microvasculature. Yet, low brain glucose levels in the mice are reversed and an overall salutary effect of the intervention is observed. In contrast, initiating protein repletion in symptomatic, adult (8-week old) mice raises brain glucose levels but fails to either mitigate brain microvasculature defects or attenuate major Glut1 DS disease features. We conclude that adequate Glut1 protein is indispensable for the proper development and maintenance of the capillary network of the brain. We further conclude that there is a limited postnatal window of opportunity to treat Glut1 DS using Glut1 augmentation as a therapeutic strategy. Nevertheless, timely reinstatement of the protein proves highly effective in preventing, indeed reversing, aspects of the disorder in the symptomatic individual.
Results
Brain microvasculature defects in Glut1 DS model mice.
Brain dysfunction is a characteristic feature of Glut1 DS patients and model mice. Moreover, the Glut1 protein is abundantly expressed in endothelia lining the brain microvasculature. We therefore began our investigation by examining the cerebral capillary network of mutant and control mice using fluorescently labelled lectin and an antibody against Glut1. As the structures identified by the two probes were invariably in perfect register (Supplementary Fig. 1a), further quantification of the microvasculature relied on lectin staining alone. This was carried out on 2-week, 8-week and 20-week old mice. We began by examining the capillaries in the thalamus, as this region appears particularly hypometabolic in positron emission tomography (PET) scans12,13,14. We found that the density of the capillaries, as determined by the total length traversed by them within a defined volume, was no different in mutants and WT mice at 2 weeks of age (Fig. 1a,b). Nor were there differences in the distribution of the sizes of individual capillaries or vessel branch points between the two cohorts of mice (Supplementary Fig. 1b,c). Brain angiogenesis continued in WT mice over the following eighteen weeks so that the capillary network had expanded by ∼27% in the thalami of 8-week animals and a further ∼5% by 20 weeks of age. In striking contrast, the capillary network of 2-week old Glut1 DS mice not only failed to expand, but rather diminished in size over the next eighteen weeks, appearing decidedly fragmented in the end. The microvasculature network in 8-week and 20-week old mutants was thus ∼30% and ∼40% respectively smaller than in age-matched controls (Fig. 1b). This diminution was not merely a consequence of a decrease in the overall extent of the capillary network, but additionally derived from smaller individual lectin-stained vessels with fewer branches (Fig. 1c,d). To ascertain if the diminished size of the capillary network in the thalami of mutants applied more generally to the cerebral microvasculature, we examined two additional regions—the cortex (primary motor and somatosensory cortex) and hippocampus (CA1, CA3 and DG regions)—of the brain. We found that the abundance of micro-vessels in these regions was similarly reduced in 20-week old mutants (capillary density: WT cortex=1,625±36, mutant cortex=1,201±32; WT hippocampus=1,092±44, mutant hippocampus=810±30, P<0.001 in each instance, t test, N≥3 mice of each genotype). Overall, the results suggest that Glut1 is required both for the elaboration as well as the maintenance of the cerebral microvasculature.