Specific Temporal Distribution and Subcellular Localization of a Functional Vesicular Nucleotide Transporter (VNUT) in Cerebellar Granule Neurons

Abstract

Adenosine triphosphate (ATP) is an important extracellular neurotransmitter that participates in several critical processes like cell differentiation, neuroprotection or axon guidance. Prior to its exocytosis, ATP must be stored in secretory vesicles, a process that is mediated by the Vesicular Nucleotide Transporter (VNUT). This transporter has been identified as the product of the SLC17A9 gene and it is prominently expressed in discrete brain areas, including the cerebellum. The main population of cerebellar neurons, the glutamatergic granule neurons, depends on purinergic signaling to trigger neuroprotective responses. However, while nucleotide receptors like P2X7 and P2Y13 are known to be involved in neuroprotection, the mechanisms that regulate ATP release in relation to such events are less clearly understood. In this work, we demonstrate that cerebellar granule cells express a functional VNUT that is involved in the regulation of ATP exocytosis. Numerous vesicles loaded with this nucleotide can be detected in these granule cells and are staining by the fluorescent ATP-marker, quinacrine. High potassium stimulation reduces quinacrine fluorescence in granule cells, indicating they release ATP via calcium dependent exocytosis. Specific subcellular markers were used to assess the localization of VNUT in granule cells, and the transporter was detected in both the axonal and somatodendritic compartments, most predominantly in the latter. However, co-localization with the specific lysosomal marker LAMP-1 indicated that VNUT can also be found in non-synaptic vesicles, such as lysosomes. Interestingly, the weak co-localization between VNUT and VGLUT1 suggests that the ATP and glutamate vesicle pools are segregated, as also observed in the cerebellar cortex. During post-natal cerebellar development, VNUT is found in granule cell precursors, co-localizing with markers of immature cells like doublecortin, suggesting that this transporter may be implicated in the initial stages of granule cell development.

New Insights into Microglia–Neuron Interactions: A Neuron’s Perspective

“…However, it is still unknown where the different motility-regulating signalling pathways intersect and how they influence each other in physiological and pathological states. Furthermore, since neurons can release ATP also during their basal activity (Ho et al, 2015;Menéndez-Méndez et al, 2017), purinergic signalling via P2Y12 receptors is essential for synaptic plasticity (Sipe et al, 2016) and microglial P2Y12 signal decreases during activation (Haynes et al, 2006), the role of purinergic signalling in physiological microglial functions cannot be ruled out and should be investigated in detail.…”

Microglia monitor and protect neuronal function via specialized somatic purinergic junctions

“…In line with this, we show that the recruitment of microglial processes to somatic junctions in the vicinity of neuronal mitochondria is linked with mitochondrial activity (NADH fluorescence) changes in neurons via P2Y12R signaling. Neurons can execute somatic ATP release via pannexin hemichannels, voltage dependent anion channels or through activity-dependent vesicle exocytosis (20,21,54). Vesicular nucleotide transporter (vNUT) is known to be responsible for somatic vesicular ATP-release in neurons (34).…”

“…Because activity-dependent exocytotic ATP or ADP release is known to take place from neuronal cell bodies under physiological conditions (20,21) and ATP (ADP) is a major chemoattractant for microglial processes via the microglial purinoceptor, P2Y12R (6,22), we next tested the hypothesis that signaling via P2Y12R is also essential for microglia-neuron interactions at these somatic junctions. In fact, microglia -but no other cells in the brain -were found to be P2Y12R-positive ( Fig.…”

Neonatal Seizures and Purinergic Signalling

“…Finally, the Cl − -dependent vesicular nucleotide transporter (VNUT) has been described to mediate the storage of ATP and other nucleotides in secretory and synaptic vesicles [106]. This transporter is highly expressed in different brain regions including the olfactory bulb, hippocampus and cerebellum [103] and has been shown to be functional in different types of neurons [102,[107][108][109] and populations of glial cells [104,105,109].…”