When Neuroligin2 sticks around, astrocytes take shape

Abstract

The complex structure of astrocytes allows for nervous system function; however, mechanisms underlying astrocyte morphogenesis remain unclear. Sakers et al find that astrocyte neuroligins (NLs) are functionally diverse, and intracellular ubiquitination of astrocyte NL2 by Nedd4l promotes astrocyte morphogenesis.

eTOC STATEMENT

Ray and Singhvi discuss recent work from Sakers and colleagues showing that Nedd4l/HECT E3 ligase ubiquitination of astrocyte NL2 promotes astrocyte morphogenesis.

Neurons transmit information within a neural circuit at contact sites called synapses, which are stabilized by intercellular adhesion. Neuroligins are conserved cell adhesion transmembrane molecules originally isolated from rat brain tissue as binding partners of Neurexins^1,2. Neurexin-neuroligin adhesion physically bridges two neurons at a synapse, enabling synaptic connectivity, and are linked to neurodevelopmental conditions like Autism². Humans express four Neuroligins (NL1-4) that function non-redundantly. NL1 primarily localizes to and modulates excitatory synapses, NL2 does so at inhibitory synapses, NL3 acts on both synapse types, and NL4 affects glycinergic synapses². Curiously, triple NL1-3 knockout animals have normal synapse number and structure, but impaired synaptic transmission, suggesting that NLs have roles beyond physically enabling neuronal synapse architecture².

Astrocytes also modulate synaptic connectivity and function at tripartite synapses through mechanisms including the secretion of neuroactive factors, ion buffering, and neurotransmitter diffusion/uptake³. This is enabled by their elaborate, bushy morphology, with fine cellular processes that contact neuronal synapses³. How astrocytes establish their ramified structures remains poorly understood molecularly. Defects in astrocyte cell shape not only affect neuronal connectivity and function, but are also correlated with genes associated with diseases like Alzheimer’s dementia³. In this issue of JCB, Sakers et al reveal insights into NL biology in astrocytes through a series of elegant molecular studies⁴. They show that, as in neurons, NL1-3 have non-redundant roles in astrocytes. By identifying their cell- and gene-specific interactomes, the authors uncover that ubiquitination of astrocytic NL2 at Lysine749 by Nedd4l/HECT ligase prevents its degradation, enabling it to drive astrocyte morphogenesis, independent of NL roles in astrocyte-driven synaptogenesis.

Rodent NL1-3, and the Drosophila ortholog, gliotactin, are expressed in both neurons and glia^2,5. Previously, the Eroglu lab showed that rodent cortical astrocytes express NL1-3 at levels approximating neurons, and loss of any NL reduces the territory size of bushy astrocytes. They also found that astrocytic NL2 binds neuronal Neurexins to promote astrocyte morphogenesis and synaptogenesis and, thereby, synapse function⁶. Building on this work, the authors aimed to systematically investigate NL redundancy and regulation in astrocytes. They first leveraged an in vitro astrocyte-neuron co-culture system to manipulate individual NLs in an astrocyte-specific manner and quantify astrocyte morphology. Depleting Nlgn2 by either shRNA or CRISPR/Cas9 mutagenesis reduced astrocyte morphological complexity. This was rescued by co-expressing a shRNA-resistant construct specifically for NL2, but not NL1 or NL3. The authors therefore concluded that the role of NL2 in regulating astrocyte morphology is distinct from NL1 or NL3.

The three NLs have similar extracellular domain (ECD) but divergent intracellular domain (ICD) sequences. In neurons, each NL-ICD has unique functions and binding partners⁷. To probe if similar biology was at play in astrocytes, Sakers et al conducted a series of structure-function studies. First, they altered known neuron-relevant ICD motifs (PDZ, poly-proline motif, gephyrin binding region) by mutagenesis but found these irrelevant for astrocyte morphology. Next, they examined chimeras of NL2-ECD fused to either the NL1 or NL3 ICD. While the chimera with NL1-ICD rescued astrocyte complexity defects of Nlgn2 depletion, that with NL3-ICD did not. They inferred that the role of NL2 in astrocyte morphogenesis required a motif shared with NL1 and absent in NL3. From bioinformatic analyses, they then identified a PPDY (PPxY) motif in the NL2 ICD. Mutating this motif (PPDY>AAAA) in either NL2 or the NL2-ECD/NL1-ICD chimera abrogated the ability of either construct to rescue morphogenesis defects of Nlgn2 knockdown, indicating that PPDY motif in NL2-ICD mediates astrocyte morphogenesis.

PPDY motifs bind Group I WW domain-containing proteins. To identify the relevant ones, the authors next performed in vivo BioID in mouse cortex for all NLs in astrocytes and NL2 in neurons. This revealed three interesting facets of NL biology. First, the astrocyte interactome of NL2 was surprisingly distinct from its known neuronal interactors as NL2 interacts with actin-binding proteins in astrocytes but not neurons. This supports past work indicating that NL2 has cell-type specific activity. Second, the identified WW domain-containing protein interactors were unique to each NL, consistent with their non-redundant astrocyte functions. Third, this dataset identified Nedd4l as a WW-domain protein that interacts with astrocytic NL2 and NL1, but not NL3.

Nedd4l is a HECT family E3 ubiquitin ligase that ubiquitin-tags astrocytic membrane proteins like Kir4.1/ K channel and Glt1/glutamate transporter for degradation^8,9. Independently, it is known that NL2 is ubiquitinated at lysine 749 (K749), although the relevance of this post-translational modification had so far been unclear¹⁰. The authors thus hypothesized that NL2 is a Nedd4l ubiquitination substrate. To test this, they first knocked down Nedd4l and found that it phenocopied NL2 depletion, but did not exacerbate it, in vitro. Nedd4l depletion similarly reduced cortical astrocyte morphological complexity in vivo. Thus, Nedd4l regulates astrocyte shape, likely in the same pathway with NL2. Interestingly, distinct from Nlgn2 loss, sparse knockdown of Nedd4l in vivo did not alter synaptogenesis. Thus, NL2 require Nedd4l-dependent stabilization to regulate astrocyte morphogenesis but not astrocyte-driven synaptogenesis.

To test the role of Nedd41 in astrocyte morphogenesis, the authors did biochemistry studies in primary rat astrocytes and HEK293T cells assays to show that the NL2 PPDY motif bound Nedd4l, and Nedd4l ubiquitinated NL2 at K749. However, post-translational modification of NL2^K749 by Nedd4l stabilized, not degraded, the protein. As a corollary, expressing the non-ubiquitin-able variant NL2^K749R in astrocyte co-cultures impaired astrocyte morphology and did not rescue NL2 knockdown defects. The authors further showed this held true also in vivo: unlike wild type NL2, expressing NL2^K749R in astrocytes did not rescue astrocyte morphology defects in the developing cortex of homozygous Nlgn2 mutant mice.

In summary, Sakers et al provide significant insight into the biology of disease-relevant NLs, particularly NL2, and developmental morphogenesis of astrocytes. It also expands the functional role of Nedd4l in astrocytes. This work also underscores the need to interrogate factors in glia and neurons with cell- and paralog-specific resolution. Finally, their shared interactome data of astrocytic NLs will be a valuable resource for future inquiry. It also raises important mechanistic questions. One, does K749 ubiquitination also stabilize neuronal NL2 or astrocytic NL1? Two, how does this developmental mechanism apply to animal aging, astrocyte heterogeneity, other glia types, or disease-state glioma cells? Finally, NLs form both homo and heterodimers². As such, how might stabilizing one NL (like NL2) alter the ratio of NL dimers an astrocyte expresses? Tackling these open questions will yield exciting insights into NL biology, astrocyte development, and synapse development and functions.

Figure 1.

Neuroligins are functionally diverse and astrocytic NL2 is ubiquitinated by Nedd4l for proper astrocyte morphogenesis. (A) The authors used in vivo iBioID to identify the interactomes for astrocyte NL1-3 and neuronal NL2. (B) In wildtype animals, Nedd4l ubiquitinates NL2 for protein stability, which allows for proper astrocyte morphology. (C) When Nedd4l is knocked down or in NL2 K749R ubiquitination mutants, astrocytes show stunted morphology. Ub = ubiquitination. Created in BioRender. Ray, S. (2026) https://BioRender.com/wkjy20h