Beyond Protein Folding: Exploring the Role of Unfolded Protein Response in Axon Targeting

Abstract

Imagine waking up to the aroma of freshly brewed coffee, nestled in a soft white blanket with early morning sunlight filtering through. The cozy scent of coffee wafts through the house, making it the perfect start to a pleasant day, especially when accompanied by a savory, buttery croissant. In that moment, one may wonder about the mechanisms underlying the ability to detect and distinguish the scents of coffee and croissants.

Odor perception is mediated by the olfactory sensory neurons (OSNs) within the nose. Each OSN contains a single type of olfactory receptor (OR) that responds to specific odorant molecules (Chess et al., 1994). Interestingly, the OSNs with the same type of receptors connect to the same micro-region within the olfactory bulb, called a glomerulus (Vassar et al., 1994). With over 1,000 different types of ORs distributed throughout the nasal cavity, the precise regulation of OSN axon targeting is crucial for odor perception.

What mechanisms enable precise axonal targeting in OSNs? The classical model suggests that each type of OR induces the expression of axon guidance molecules in an activity-dependent manner, sorting the OSN axons to their respective glomeruli (Chen and Flanagan, 2006). However, several studies have revealed that axonal targeting is not entirely disrupted even in the absence of odor-evoked neural activity, indicating an activity-independent mechanism (Belluscio et al., 1998; Lin et al., 2000). Emerging research by Shayya et al. (2022) suggests a potential role in the relationship between ORs and ER stress in regulating the OSN axon targeting.

Shayya et al. (2022) generated a translational fluorescent reporter for ER stress by replacing the Atf5 coding sequence with iRFPp2a-Cre, which enables the fluorescence of the near-infrared fluorescent protein (iRFP) to quantify the unfolded protein response (UPR). By measuring the iRFP levels in the different OSN types tagged with iresGFP, they discovered the distinct levels of ER stress among the different OSN types, with M71 > Mor23 > Mor28 > P2 > Class1. A swap experiment, where the moderate-stress OR protein P2 was replaced with the high-stress OR protein M71, revealed significantly higher stress levels in the M71 → P2 “swap” OSNs than in the endogenous P2 OSNs. Multiple sequence alignment of all OR proteins showed that the ORs with similar sequences had similar levels of ER stress, thus further confirming that the OR sequence determines the ER stress levels.

The mapping of the ORs to their corresponding levels of ER stress was categorized as either high or low stress. A comparison between the differentially expressed genes of these groups demonstrated that the expression of axon guidance molecules was dependent on the ER stress level. To assess whether this relationship between ER stress and axon guidance gene expression affected the specificity of OSN axon targeting, researchers employed a specialized approach. They used a monoallelic deprivation strategy by generating OR (iresCre/iresGFP); Rosa26 (LSL-tdTomato/+) mice for the M71 and Mor28 OR proteins. This strategy utilized the monoallelic expression of a specific OR gene, where the OSNs expressing the OR can either choose the iresGFP allele (serving as an internal control for GFP expression) or the iresCre-tagged allele (allowing for recombination of the floxed alleles and marked by tdTomato). To study the effect of ER stress on axon guidance, these mice were crossed with Perk-floxed mice, generating a Cre-expressing tdTomato allele that could adopt one of three variations: wild-type (WT), Perk heterozygous knockout, and Perk conditional knockout (cKO).

In WT Perk mice, the red and green axons target the same glomeruli in the olfactory bulb. However, in Perk cKO M71 OSNs, which experience a significant reduction in the UPR, the red axon loses glomerular coalescence. They stretch out without coming together into a specific glomerulus, resulting in a disorganized meshwork. In contrast, in the Mor28 Perk heterozygous knockout mice, which experience a slight reduction in the UPR, red axon targeting was slightly shifted without disrupting glomerular coalescence.

When one allele of Hspa5, an ER chaperone that reduces PERK signaling, was removed to increase the ER stress, the red and green axons exhibited segregation, innervating different glomeruli. Given that axon guidance responds to both the decrease and increase in ER stress, the authors propose that OSNs have two distinct PERK-dependent regulatory networks with different saturation thresholds: a “glomerular coalescence” network activated at lower UPR levels and an “axon guidance” network activated at higher UPR levels, which controls the targeting specificity.

The authors examined the UPR effectors in the axon guidance network by analyzing single-cell RNA-sequencing data for differentially expressed master regulator transcription factors (mrTFs) between the high and low ER-stress ORs. They identified Ddit3, a UPR-responsive TF, as the leading mrTF candidate in high-stress OSNs. Indeed, the removal of Ddit3 altered the M71 and Mor28 OSN axon targeting, resulting in shifted glomerular positions without affecting axonal coalescence. Expanding the Ddit3 deletion study to include all mOSNs revealed a significant reduction in the expression of nine axon guidance molecules, while the ORs and most other genes remained unchanged. The high-stress axon guidance molecule set was the most significantly depleted gene set in Ddit3 cKO versus WT mOSNs. Additionally, intermediate levels of axon guidance molecules were observed in heterozygous Ddit3 deletion, indicating that ER stress levels function as a “rheostat” to control the OSN axon guidance.

The significance of this finding lies in that the canonical stress response pathway plays a crucial role in converting OSN neuronal identity into axon targeting. Although the UPR has traditionally been linked to protein-folding stress (Schroder and Kaufman, 2005), recent studies have revealed that it has additional vital functions, including lipid metabolism, energy regulation, inflammation, and cell fate (Rutkowski and Hegde, 2010). This study extends the conventional role of UPR, demonstrating its potential to facilitate neural circuit wiring.

Furthermore, this alternative pathway provides insight into how the olfactory bulb maintains a stable glomerular map in an unpredictable environment. Previously, it was believed that odor-evoked activity patterns mediate the expression of axon guidance molecules in glomerular sorting (Chen and Flanagan, 2006; Nakashima, et al., 2019). However, it remains unclear how the various ORs receive stable sensory inputs for the neural circuit formation. This study proposes an alternative signaling pathway that utilizes many of the same axon guidance molecules to map the OR identity, even in the absence of olfactory sensory input. This study suggests that PERK detects subtle amino acid changes in many ORs, thus enabling ER stress to tile the OR proteins. The complete disruption of glomerular coalescence upon the deletion or saturation of PERK implies the significant role of ER stress in achieving the precise wiring required for the olfactory systems, thereby overshadowing odor-evoked activity.

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