Elsevier

Experimental Eye Research

Volume 171, June 2018, Pages 54-61
Experimental Eye Research

Role of SARM1 and DR6 in retinal ganglion cell axonal and somal degeneration following axonal injury

https://doi.org/10.1016/j.exer.2018.03.007Get rights and content

Highlights

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    SARM1, but not DR6, deficiency delays distal axonal degeneration in RGCs.

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    Neither SARM1 nor DR6 deficiency increases RGC survival following optic nerve crush.

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    DLK/JNK signaling in RGC somas is unaffected by genetic disruption of SARM1.

Abstract

Optic neuropathies such as glaucoma are characterized by the degeneration of retinal ganglion cells (RGCs) and the irreversible loss of vision. In these diseases, focal axon injury triggers a propagating axon degeneration and, eventually, cell death. Previous work by us and others identified dual leucine zipper kinase (DLK) and JUN N-terminal kinase (JNK) as key mediators of somal cell death signaling in RGCs following axonal injury. Moreover, others have shown that activation of the DLK/JNK pathway contributes to distal axonal degeneration in some neuronal subtypes and that this activation is dependent on the adaptor protein, sterile alpha and TIR motif containing 1 (SARM1). Given that SARM1 acts upstream of DLK/JNK signaling in axon degeneration, we tested whether SARM1 plays a similar role in RGC somal apoptosis in response to optic nerve injury. Using the mouse optic nerve crush (ONC) model, our results show that SARM1 is critical for RGC axonal degeneration and that axons rescued by SARM1 deficiency are electrophysiologically active. Genetic deletion of SARM1 did not, however, prevent DLK/JNK pathway activation in RGC somas nor did it prevent or delay RGC cell death. These results highlight the importance of SARM1 in RGC axon degeneration and suggest that somal activation of the DLK/JNK pathway is activated by an as-yet-unidentified SARM1-independent signal.

Introduction

Glaucoma is an optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs), projection neurons that are responsible for processing and transmitting visual information to the brain. A key early pathophysiological event is the injury of RGC axons at the lamina cribrosa of the optic nerve head. In order to study axon injury signaling, RGC axons can be experimentally severed in the optic nerve crush (ONC) model. In response to axotomy, RGC axons degenerate and cell death ensues. It is known that this cell death program uses a multi-tiered somal signaling cascade involving dual leucine zipper kinase (DLK) and leucine zipper kinase (LZK), mitogen-activated protein kinase (MAPK) kinases 4 and 7 (MKK4/MKK7), JUN N-terminal kinases 1ā€“3 (JNK1-3), JUN and activating transcription factor 2 (ATF2) and, ultimately, BAX (Fernandes et al., 2012, 2014; Li et al., 2000; Libby et al., 2005; Watkins et al., 2013; Welsbie et al., 2013, 2017). Interestingly, the role of other BH3 family members like PUMA/BBC3 is more enigmatic with in vitro data supporting a role for PUMA downstream of DLK signaling, but with Puma-null RGCs showing little protection from ONC in vivo (Harder and Libby, 2011b, 2013; Simon et al., 2016).

The pathways responsible for cell death partially overlap with those signaling cascades responsible for axonal degeneration. For example, inhibition of MAPK signaling or BH3 proteins like BAX and PUMA delays axon degeneration in various neuronal cell types (Howell et al., 2007; Libby et al., 2005; Nikolaev et al., 2009; Simon et al., 2016; Yang et al., 2015). DLK may also play a minor role in axon degeneration in some models of axon injury (Ghosh et al., 2011; Miller et al., 2009), although we have previously shown that targeted disruption of Dlk did not affect distal axon degeneration in the ONC model (Fernandes et al., 2014). Two of the most robust mediators of axon degeneration appear to be sterile alpha and TIR motif containing 1 (SARM1), a toll-like receptor adaptor family member, and death receptor 6 (DR6), a tumor necrosis factor receptor superfamily member (Gamage et al., 2017; Gerdts et al., 2013; Nikolaev et al., 2009; Osterloh et al., 2012).

SARM1 was initially identified in a forward genetic screen for Drosophila mutants that showed long-term axon survival after axotomy (Osterloh et al., 2012), suggesting SARM1 has a role in axonal degeneration. SARM1 is found in axons and is necessary for axonal degeneration in multiple neuronal cell types and in response to a wide range of axonal insults (Geisler et al., 2016; Osterloh et al., 2012; Turkiew et al., 2017). Forcing SARM1 multimerization is sufficient to activate MAPK signaling and induce axonal degeneration, suggesting that SARM1 functions upstream of the MAPK pathway (Gerdts et al., 2013; Yang et al., 2015). Consistent with this hypothesis, inhibition of MAPK signaling reverses the SARM1-dependent effect on hippocampal neuron dendritic complexity (Chen et al., 2011). However, others have recently shown that MAPK signaling modulates SARM1 activity by degrading the SARM1 inhibitor, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), potentially placing SARM1 downstream of MAPK signaling. Moreover, SARM1 can directly consume the axon survival-promoting molecule, nicotinamide adenine dinucleotide (NAD+) (Gerdts et al., 2015 and Essuman et al., 2017), further supporting a role for SARM1 as a downstream effector. In retina, SARM1 mRNA and protein are selectively expressed by RGCs and genetic disruption of Sarm1 delays the degeneration of RGC axons following direct axotomy or an excitotoxic challenge (Yang et al., 2015, Massoll et al., 2013). Interestingly, several studies have also demonstrated a role for SARM1 in promoting neuronal cell death. SARM1 deficiency has been shown to reduce neuronal cell death induced by oxygen-glucose deprivation (Kim et al., 2007) and viral infection (Mukherjee et al., 2013). Additionally, SARM1 gain of function mutants have been shown to cause neuronal cell death (Gerdts et al., 2013; Yang et al., 2015). Recently, SARM1 was shown to be critical for Sarmaptosis, a novel form of cell death induced by mitochondrial dysfunction (Summers et al., 2014). Given the overlap between axon degeneration and cell death pathways, the unclear relationship between MAPK signaling and SARM1 and the known role of SARM1 in RGC axon degeneration, it is important to investigate whether SARM1 is necessary for the MAPK activation and cell death that follow axon injury.

DR6 (TNFRSF21) is another molecule that has been shown to be involved in axonal degeneration. It was initially identified to be critical for axonal degeneration of dorsal root ganglion neurons following trophic-factor deprivation (Nikolaev et al., 2009). Cleaved Ī²-amyloid precursor protein (APP) was shown to activate DR6 and trigger axonal degeneration by activating a downstream caspase signaling cascade (Nikolaev et al., 2009). Collectively, these previous studies provide strong support for SARM1 and DR6 as critical regulators of axon degeneration, making them attractive targets to assess in the context of glaucomatous axonal degeneration. In this report, we use Sarm1 and DR6 knockout animals to explore the role of these genes in RGC somal and axonal degeneration.

Section snippets

Mice

Mice carrying a null allele of Sarm1 (B6.129X1-Sarm1tm1Aidi/J) were obtained from the Jackson Laboratories (Stock Number: 018069). The Wallerian Degeneration Slow (WldS) allele (Lunn et al., 1989; Mack et al., 2001) was backcrossed into C57BL/6Jā€Æ>ā€Æ20 generations. DR6āˆ’/āˆ’ mice (Tnfrsf21tm2Gne) were obtained from Genentech (Zhao et al., 2001). To genetically label RGC axons for histological assessment of morphological signs of axon degeneration, Thy1-CFP mice (B6.Cg-Tg(Thy1-CFP)23Jrs/J, Stock

SARM1 is not required for RGC somal degeneration after axonal injury

In order to explore whether SARM1 has a role in RGC cell death, we first tested whether targeted deletion of Sarm1 increased the survival of primary RGCs isolated from Sarm1-null mice as compared to WT controls. We have previously demonstrated that the activity of RGC-intrinsic genes in this model is highly correlated with activity in vivo in the mouse ONC model (Welsbie et al., 2013, 2017). Primary RGCs were isolated from P0-P3 WT or Sarm1-null mice, challenged with 1ā€ÆĪ¼M colchicine at 48ā€Æh and

Distinct signaling cascades for somal and axonal degeneration

Our data support previous findings by Osterloh et al., and showed that SARM1 deficiency was as protective as WldS in preventing axon degeneration. However, sarmaptosis does not contribute to RGC death after mechanical axon injury. Collectively, these data show that SARM1 is critical for axonal but not somal degeneration of RGCs after ONC. These results are consistent with previous studies that suggest that distinct signaling pathways control the degeneration of different cellular compartments

Conclusion

After mechanical axonal injury (ONC) the degeneration pathways controlling somal and axonal degeneration appear to be molecularly distinct (Fig. 7). Proximal to the site of injury, DLK/LZK, JNK2/3 and JUN dependent MAPK signaling have been shown to be important for a BAX-dependent RGC somal degeneration after ONC (Fernandes et al., 2012, 2014; Li et al., 2000; Libby et al., 2005; Watkins et al., 2013; Welsbie et al., 2013, 2017). Moreover, ER stress and other factors are also important

Acknowledgements

The authors would like to thank Aaron DiAntonio and Genentech for generously providing the Sarm1 and Dr6 mice, respectively. This work was supported by The Glaucoma Research Foundation (RTL), The E. Matilda Ziegler Foundation, Research to Prevent Blindness, Guerrieri Family Foundation, EY018606 (RTL), EY023754 (DJZ), and Research to Prevent Blindness unrestricted grants to the Departments of Ophthalmology at the University of Rochester Medical Center, University of California, San Diego and the

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