Suppression of guanylyl cyclase (β1 subunit) expression impairs neurite outgrowth and synapse maturation in cultured cerebellar granule cells

Abstract

The increased expression of different soluble guanylyl cyclase (sGC) subunits during development is consistent with these proteins participating in the formation and establishment of interneuronal contacts. Functional sGC is generated by the dimerization of an a-subunit (sGCa1/2) with the b1-subunit (sGCb1), and both depletion of the sGCb1 subunit and inhibiting sGC activity impair neurite outgrowth. Similarly, impairing sGC activity diminishes the amount of growth-associated protein (GAP-43) and synapsin I, two proteins that participate in axon elongation and synaptogenesis, suggesting a role for sGC in these processes. Indeed, fewer synapses form when sGC is inhibited, as witnessed by FM1-43 imaging and synapsin I immunostaining, and the majority of synapses that do form remain functionally immature. These findings highlight the importance of sGC in the regulation of neurite outgrowth and synapse formation, and in the functional maturation of cerebellar granule cells in vitro. During neuronal development, cGMP signaling is important to modulate growth cone activity in a variety of cell types. [1][2][3] Indeed, intracellular cGMP synthesis is linked to and mediates the neurogenic effects of different factors, including that of nerve growth factor (NGF). 4 The expression of the different guanylyl cyclase subunits (sGC), the main physiological receptor for nitric oxide (NO), augments throughout development in different areas of the rat brain and in in vitro models of neuronal maturation. 5-7 Accordingly, these proteins may be important for the formation and growth of neuronal processes, as well as for the establishment of intercellular contacts and the refinement of functional neuronal connections within the peripheral and central nervous system. 1,8,9 There are two known isoforms of sGC, the ubiquitous a 1 b 1 and a 2 b 1 , which has a more limited distribution. 10 This latter isoform interacts with synaptic scaffolding proteins through its PDZ domains, suggesting that this enzyme is accumulated in synapses 11,12 and that it is responsible for cGMP synthesis in this cellular compartment. 13 GAP-43 is an integral membrane protein associated with the cytoplasmic surface of axonal growth cones in developing neurons, and it is generally considered an intrinsic determinant of neurite outgrowth and plasticity. 14,15 In addition, GAP-43 also plays important roles in synaptogenesis as well as in regulating the cytoskeletal organization of the nerve ending. 16 Indeed, neurite outgrowth and axonal pathfinding are affected when GAP-43 expression is altered, both during development and in cultured cells. Likewise, synapsins are also thought to participate in the processes of axon elongation and synapse formation during development, and the expression of synapsin I correlates well with synapse maturation. 17 Synapsins are exclusively associated with small synaptic vesicles and they are virtually excluded from other tissues or neuroendocrine cells. 18 Indeed, synapsin I is probably the most specific marker of synapses in the ...

Nos2 Inactivation Promotes the Development of Medulloblastoma in Ptch1+/− Mice by Deregulation of Gap43–Dependent Granule Cell Precursor Migration

“…Other studies already suggested a link between Gap43 mRNA levels and NO signaling due to co-induction of nNos and Gap43 during axon regeneration and reactive synaptogenesis following injury of spinal motoneurons [49] and sensory neurons [50]. Furthermore, a downregulation of Gap43 was reported after silencing of soluble guanylate cyclase subunits, the central elements of cGMP-mediated NO signaling [51]. Finally, the present study demonstrates Gap43 downregulation to be a consequence of NO withdrawal in neuronal progenitors and MB cells.…”

Cannabinoid Type 1 Receptors Transiently Silence Glutamatergic Nerve Terminals of Cultured Cerebellar Granule Cells

“…The cell suspension was electroporated with Amaxa™ Nucleofector II using Rat Neuron Amaxa Nucleofector Kit (Lonza), following the instructions provided by the manufacturer (programme O-03) as described [34]. For transfection, 1 µg of VGLUT1-pHluorin (VGpH) DNA [27] was used per 5×10 6 cells and the cells were finally seeded at a density of 1.6×10 6 cells/coverslip.…”

The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

“…In this sense, both 8-Br-cGMP and NMDA induce an increase in the density of synapsin puncta [30] and GluA1 clusters, effects that are counteracted and reversed by KT5823. The increase in cluster density was accompanied by an increase in immunofluorescence intensity compatible with the presence of more protein in these clusters, while conversely, the reduced cluster density provoked by exposure to KT5823 was accompanied by a reduction in their mean fluorescence.…”

“…In culture, cerebellar granule cells establish synapses with themselves and their development is also affected by the NO and cGMP pathway. While NO negatively regulates precursor proliferation through a cGMP-independent mechanism [65], it also triggers differentiation and maturation by a cGMP-dependent one [30,31,66]. …”

“…For the siRNA experiments (see Figure 6), the ROIs (regions of interest) were selected with Igor Pro software having established standard parameters to select only specific antibody stained clusters. The automatic threshold with the Isodata algorithm [43] was then used to convert the image into a binary mask that included all the fluorescence emissions above background, and spurious ROIs were removed manually [30]. The staining intensities were then normalized to the MAP2 labelling (internal control for quantitative analysis) and used to compare signal intensities between the control and treated cells.…”