Trafficking and Fusion of Neuropeptide Y-Containing Dense-Core Granules in Astrocytes

Abstract

It is becoming clear that astrocytes are active participants in synaptic functioning and exhibit properties, such as the secretion of classical transmitters, previously thought to be exclusively neuronal. Whether these similarities extend to the release of neuropeptides, the other major class of transmitters, is less clear. Here we show that cortical astrocytes can synthesize both native and foreign neuropeptides and can secrete them in a stimulation-dependent manner. Reverse transcription-PCR and mass spectrometry indicate that cortical astrocytes contain neuropeptide Y (NPY), a widespread neuronal transmitter. Immunocytochemical studies reveal NPY-immunoreactive (IR) puncta that colocalize with markers of the regulated secretory pathway. These NPY-IR puncta are distinct from the synaptic-like vesicles that contain classical transmitters, and the two types of organelles are differentially distributed. After activation of metabotropic glutamate receptors and the release of calcium from intracellular stores, the NPY-IR puncta fuse with the cell membrane, and the peptidecontaining dense cores are displayed. To determine whether peptide secretion subsequently occurred, exocytosis was monitored from astrocytes expressing NPY-red fluorescent protein (RFP). In live cells, after activation of glutamate receptors, the intensity of the NPY-RFP-labeled puncta declined in a step-like manner indicating a regulated release of the granular contents. Because NPY is a widespread and potent regulator of synaptic transmission, these results suggest that astrocytes could play a role in the peptidergic modulation of synaptic signaling in the CNS.

Excitable Astrocytes: Ca2+- and cAMP-Regulated Exocytosis

“…DCVs in cultured astrocytes contain the secretory proteins secretogranins II [34,65,103] and III [104]. Besides secretogranins, DCVs/less DCVs [28] in culture can also store chromogranins [42], ANP [19,65], neuropeptide Y (NPY) [103,105] and a fraction of cellular ATP [36,37]. DCVs containing secretogranins were recently reported in astrocytes in human brain tissue [42], confirming the existence of DCV vesicles also in situ.…”

“…DCVs containing secretogranins were recently reported in astrocytes in human brain tissue [42], confirming the existence of DCV vesicles also in situ. DCVs are typically larger than SLMVs (*100-600 nm) [34,42,103] and do not apparently co-localize with VAMP2 and vGLUT1 in culture, indicating that they belong to a distinct vesicle population like SLMVs [65,105]. DCV gliosignal molecules are discharged from astrocytes upon stimulation; whether they are co-released from the same DCVs or belong to distinct subpopulations of DCVs still needs to be investigated.…”

TI‐VAMP/VAMP7 is the SNARE of secretory lysosomes contributing to ATP secretion from astrocytes

“…Two main mechanisms of release of small transmitters have been defined: (i) a direct loss of the chemical transmitters from the cytoplasm into the extracellular medium, mediated by anion channels, connexin hemichannels, P2X 7 receptor channels and transporter proteins, which is favoured in conditions such as ischemia, traumatic injury and seizure activity (Evanko et al, 2004;Hamilton and Attwell, 2010) and (ii) a regulated form of exocytosis, mediated by clear vesicles, with a diameter of 30-100 nm, deputed to the release of glutamate and D-serine (Bezzi et al, 2004;Crippa et al, 2006;Mothet et al, 2005;Zhang et al, 2004), large dense core vesicles, which store secretogranin II (Sg II) (Calegari et al, 1999;Hur et al, 2010) peptides (ANP) and a fraction of cellular ATP (Coco et al, 2003;Pangrsic et al, 2007;Parpura and Zorec, 2010;Pascual et al, 2005;Pryazhnikov and Khiroug, 2008;Ramamoorthy and Whim, 2008;Striedinger et al, 2007) and secretory lysosomes (Jaiswal et al, 2007;Li et al, 2008;Zhang et al, 2007) which contain ATP but not glutamate. On the basis of live cell imaging, it was recently proposed that lysosomes are the major vesicular compartment undergoing calciumregulated exocytosis from cortical astrocytes (Li et al, 2008).…”

PKC‐dependent inhibition of CA2+‐dependent exocytosis from astrocytes

“…Astrocytes express a variety of neurotransmitter receptors (Kettenmann and Steinh€ auser, 2005) and respond to neuronal activity by increasing intracellular calcium levels and producing second messengers (Agulhon et al, 2008). Furthermore, astrocytes release neurotransmitters such as glutamate and ATP (Coco et al, 2003;Pasti et al, 2001) and many bioactive peptides including growth factors and cytokines (Kržan et al, 2003;Ramamoorthy and Whim, 2008) by Ca 21 -dependent exocytosis. Thus, it is widely believed that astrocytes perform additional important functions in the brain, and may regulate the activity of synapses and neural networks through bidirectional interactions with neurons (Araque et al, 1999;Pascual et al, 2005;Perea and Araque, 2007).…”