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

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

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

<i>cul3</i> and <i>kel</i> interact genetically, mutations in <i>kel</i> rescue the <i>cul3</i>-mutant phenotype.

<p>Quantification of dendritic ends in wild type, <i>cul3^gft2</i>, <i>kel^DE1</i>, and <i>cul3^gft2 kel^DE1</i> double-mutant clones in ddaA (A), ddaB (B), ddaC (C), ddaF (D), ddaE (E), and ddaD (F) MARCM neurons. Note: small protrusions were not counted in <i>cul3</i>-mutant ddaE and ddaD neurons. Decreasing levels of Kelch in <i>cul3^gft2 kel^DE1</i> double-mutant single-cell clones partially or completely rescued the <i>cul3</i>-mutant phenotype. ***: p<0.001, **: p<0.01.</p

Kelch levels are critical for dendritic branching, and regulated by COP9^Cullin3.

<p>(A) Western blot showing that Kelch is accumulated in <i>cul3</i> mutants. α-Tubulin was used as a loading control. (B, C) Kelch over-accumulates in <i>cul3</i> and <i>CSN5</i>-mutant neurons, and is virtually undetectable in the adjacent non-mutant neurons. (B′) and (C′) show Kelch (red) staining only from the panels (B) and (C). Cell bodies are marked by arrows, accumulation of Kelch at the terminal branches is pointed by arrowheads, magnified parts are marked by the dotted boxes. GFP is green, Kelch is red. (D, D′) A <i>cul3</i>-mutant epithelial cell with clumps of Kelch. (E) Kelch and Cullin3 interact physically. Complex containing the Cullin3-GST fusion protein was pulled down from wt or <i>cul3</i>-mutant larvae lysates. Presence of Kelch and GST-Cullin3 fused protein in the complex was determined by anti-Kelch and anti-GST, respectively. (F) A ddaC neuron visualized by the <i>477-GAL4</i>-driven expression of <i>UAS-GFP</i>. (G) Expression of <i>UAS-Kelch</i> under the control of <i>477-GAL4</i> stimulates dendritic branching. (H) In <i>kel</i> mutants, ddaC dendritic tree is simpler.</p

Overaccumulation of Ci inhibits dendritic development.

<p>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>109(2)80-GAL4</i>-driven overexpression of Ci, one of the Cullin1^Slimb targets, leads to reduced dendritic branching. (C, E) Ci is over-accumulated in <i>CSN5</i>- or <i>Nedd8</i>-mutant neurons with severely repressed dendritic branching. (D, F) Levels of Ci are lower in <i>CSN5</i>- or <i>Nedd8</i>-mutant neurons without the strong reduction of elaboration. (G) Ci is over-accumulated in <i>slmb</i> MARCM clones. (H) Levels of Patched, one of the Hh targets, are elevated in <i>slmb</i>-mutant neurons. Scale bar: 50 µm.</p

<i>cul3</i>-mutant neurons show small protrusions; Cullin3 overexpression represses dendritic development.

<p>(A, C) Typical wild type ddaE and ddaD neurons. (A′) and (C′) are magnified images from panels (A) and (C). (B, D) <i>cul3</i>-mutant ddaE and ddaD neurons. (B′) and (D′) are magnified images from panels (B) and (D). In <i>cul3</i> mutants, normally smooth ddaE and ddaD neurons generate small protrusions. (E) A wild type epithelial cell. (F) <i>cul3</i>-mutant epithelial cells with multiple protrusions. (G) PNS neurons in a non-mutant third instar larva, visualized by the <i>109(2)80-GAL4</i>-driven expression of GFP. (H) Overexpression of Cullin3 leads to repression of dendritic branching.</p

CSN acts in neurons via Cullin1 or Cullin3.

<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>cullin1</i> interact genetically, simultaneous reduction of their functions represses dendritic development. (C) <i>CSN5</i> and <i>cullin3</i> interact genetically, <i>CSN5/cul3</i> double heterozygotes show increased dendritic elaboration. Single <i>CSN5, cullin1</i> or <i>cul3</i> heterozygous have no effect on dendritic development (not shown) (D) MARCM clone of a wild type ddaC neuron. (E) Loss of <i>cullin1</i> lead to decreased branching. (F) <i>cullin3</i>-mutant ddaC neurons demonstrate abnormal over-branching phenotype. Scale bar: 50 µm.</p

Reduction of <i>CSN5</i> alters neuronal development.

<p>(A) PNS dendritic arborization neurons in a non-mutant third instar larva, visualized by the <i>109(2)80-GAL4</i>-driven expression of UAS-GFP. (B) <i>CSN5^N</i> heterozygotes have normal dendritic arborization. (C) <i>CSN5^N</i>/<i>CSN5^quo1</i> trans-heterozygotes and (D) <i>CSN5^N</i> homozygotes repress dendritic branching. In all figures anterior is oriented to the left. Scale bar: 50 µm.</p