Dual Regulation of Dendritic Morphogenesis in Drosophila by the COP9 Signalosome

Altered dendritic arborization contributes to numerous physiological processes including synaptic plasticity, behavior, learning and memory, and is one of the most consistent neuropathologic conditions found in a number of mental retardation disorders, schizophrenia, and neurodegenerative disease. COP9 signalosome (CSN), an evolutionarily conserved regulator of the Cullin-based ubiquitin ligases that act in the proteasome pathway, has been found associated with diverse debilitating syndromes, suggesting that CSN may be involved in regulation of dendritic arborization. However, the mechanism of this control, if it exists, is unknown. To address whether the CSN pathway plays a role in dendrites, we used a simple and genetically tractable model, Drosophila larval peripheral nervous system. Our model study identified the COP9 signalosome as the key and multilayer regulator of dendritic arborization. CSN is responsible for shaping the entire dendritic tree through both stimulating and then repressing dendritic branching. We identified that CSN exerts its dualistic function via control of different Cullins. In particular, CSN stimulates dendritic branching through Cullin1, and inhibits it via control of Cullin3 function. We also identified that Cullin1 acts in neurons with the substrate-specific F-box protein Slimb to target the Cubitus interruptus protein for degradation

COP9 Limits Dendritic Branching via Cullin3-Dependent Degradation of the Actin-Crosslinking BTB-Domain Protein Kelch

Components of the COP9 signalosome (CSN), a key member of the conserved 26S proteasome degradation pathway, have been detected to be altered in patients of several debilitating syndromes. These findings suggest that CSN acts in neural circuits, but the exact function of CSN in brain remains unidentified. Previously, using Drosophila peripheral nervous system (PNS) as a model system, we determined that CSN is a critical regulator of dendritic morphogenesis. We found that defects in CSN led to the strikingly contrast phenotype of either reducing or stimulating dendritic branching. In particular, we have reported that CSN stimulates dendritic branching via Cullin1-mediated proteolysis. Here we describe that CSN inhibits dendritic arborization in PNS neurons acting via control of Cullin3 function: loss of Cullin3 causes excessive dendritic branching. We also identified a downstream target for Cullin3-dependent degradation in neurons – the actin-crosslinking BTB-domain protein Kelch. Inappropriate accumulation of Kelch, either due to the impaired Cullin3-dependent turnover, or ectopic expression of Kelch, leads to uncontrolled dendritic branching. These findings indicate that the CSN pathway modulates neuronal network in a multilayer manner, providing the foundation for new insight into the CSN role in human mental retardation disorders and neurodegenerative disease

Quantification of total dendritic area of ddaC neurons.

<p>Quantification of total dendritic area of ddaC neurons.</p

Cullin1 and Cullin3 have opposite effects on dendritic arborization.

<p>(A) Average number of the terminal dendritic ends in wild type, <i>cul1</i> and <i>cul3</i> mutant ddaC neurons. (B) Quantifications of total dendritic length in wild type, <i>cul1</i> and <i>cul3</i> mutant ddaC neurons. (C) Quantifications of total dendritic area in wild type, <i>cul1</i> and <i>cul3</i> mutant ddaC neurons. (D) Sholl analysis histogram of dendritic arbors of wt, <i>cul1</i> and <i>cul3</i> ddaC clones. Error bars represent standard deviation.</p

Reversed Strahler analysis.

<p>Values are the mean (± standard deviation) number of total dendritic branches in each order. The total number of neurons observed is indicated in parentheses. “–” indicates order that was not observed for the particular genotype.</p

Kelch stimulates dendritic elaboration in a Cullin3-dependent manner.

<p>(A, F, K, P) MARCM clones of wild type ddaC, ddaA, ddaB, ddaF neurons, respectively. (B, G, L, Q) MARCM clones of <i>UAS-Kelch</i> in these types of neurons, Kelch overexpression stimulates dendritic branching. (C, H, M, R) Double clones of <i>cul3</i> and <i>UAS-Kelch</i> in ddaC, ddaA, ddaB, ddaF neurons. Simultaneous Kelch overexpression and loss of Cullin3 function resulted in increased branching phenotypes, especially in ddaB (M) and ddaF (R) neurons. Scale bar: 50 µm. (D, I, N, S) Quantifications of terminal dendritic ends in wild type, <i>UAS-Kelch</i>, and <i>cul3 UAS-Kelch</i> double clones in ddaC, ddaA, ddaB, ddaF neurons, respectively. (E, J, O, T) Quantifications of total dendritic length in wild type, <i>UAS-Kelch</i>, and <i>cul3 UAS-Kelch</i> double clones in ddaC, ddaA, ddaB, ddaF neurons, respectively. ***: p<0.001, **: p<0.01, *: p<0.02.</p

Reversed Strahler analysis of the ddaC neurons.

<p>Values are the mean (± standard deviation) number of total dendritic branches in each order. The total number of neurons observed is indicated in parentheses. “–” indicates order that was not observed for the particular genotype.</p

Loss of <i>cullin3</i> stimulates dendritic elaboration.

<p>(A, E, I, M) MARCM clones of wild type ddaC, ddaA, ddaB, ddaF neurons, respectively. (I′) and (M′) are magnified images from panels (I) and (M). In contrast to wild type, <i>cul3</i>-mutant ddaC (B), ddA (F), ddaB (J), ddaF (N) neurons show increasing dendritic branching. (J′) and (N′) are magnified images from panels (J) and (N). Scale bar: 50 µm. (C, G, K, O) Quantifications of terminal dendritic ends in wild type and two independent <i>cul3</i> alleles mutant ddaC, ddaA, ddaB, ddaF neurons, respectively. (D, H, L, P) Quantifications of total dendritic length in wild type and <i>cul3^gft2</i> mutant ddaC, ddaA, ddaB, ddaF neurons, respectively. ***: p<0.001, **: p<0.01, *: p<0.02.</p

Stabilization of Kelch promotes dendritic protrusions.

<p>(A, C) Wild type ddaE and ddaD neurons. (B, D) ddaE and ddaD neurons of <i>cul3 UAS-Kelch</i> double clones. (A′), (B′), (C′) and (D′) are magnified fragments of panels (A), (B), (C) and (D). Impaired degradation of Kelch expression led to the second level protrusions in normally smooth ddaE and ddaD neurons.</p

The F-box protein Slimb is involved in dendritic branching.

<p>(A) PNS neurons in a non-mutant third instar larva, visualized by the <i>109(2)80-GAL4</i>-driven expression of <i>UAS-GFP</i>. (B) <i>CSN5</i> and <i>slmb</i>, a component of the SCF complex, interact genetically in dendritic development leading to reduced dendritic branching. (C, F, I) Typical wild type ddaC, ddaF, ddaB neurons, respectively. (D, G, J) <i>slmb</i>-mutant mosaic clones of ddaC, ddaF, ddaB neurons with the characteristic fewer branching phenotype. Scale bar: 50 µm. (E, H, K) Quantifications of terminal dendritic ends in wild type and <i>slmb</i> mutant ddaC, ddaF and ddaB neurons. ***: p<0.001, **: p<0.01, *: p<0.03.</p