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

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

Kimberly A Fernandes et al. Exp Eye Res. 2018 Jun.

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.

Keywords: Axon degeneration; Dual leucine zipper kinase (DLK); Neurodegenerative disease; Retinal ganglion cell (RGC); Sterile alpha and TIR motif containing 1 (SARM1).

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Figures

Figure 1
Figure 1. Targeted disruption of SARM1 does not prevent primary RGC cell death

A. CellTiter Glo assay (±SD; n = 4 per sample) of primary RGCs isolated from WT and Sarm1-null mice, treated with either vehicle or the DLK/LZK inhibitor tozasertib (1 μM), 48 hours after a challenge with 1 μM colchicine. As opposed to DLK/LZK inhibition, loss of SARM1 activity did not increase survival of RGCs in vitro. RLU, relative luciferase units. B. CellTiter Glo assay (±SD; n = 4 per sample) of primary RGCs isolated from SpCas9-expressing mice and transfected with either of two gRNAs targeting Sarm1, a 50:50 mix of gRNAs targeting Dlk/Lzk or a non-targeting control, 48 hours after a challenge with 1 μM colchicine.

Figure 2
Figure 2. Neither SARM1 deficiency or WldS expression alter RGC death after ONC

A. Cell counts of cleaved caspase 3+ (cCASP3+) cells 5 days after ONC. Neither Sarm1 deficiency (Sarm1+/− or Sarm1−/−) nor WldS expression altered the number of dying RGCs compared to WT mice (n ≥ 6, All genotypes; P>0.05 for all comparisons). Note, cCasp3+ cells were not found in uninjured mice of any genotype (n ≥ 4 for each genotype). B. Representative images of flat mounted retinas labeled with TUJ1 from WT, Sarm1−/− and WldS mice. C. Cell counts of the number of TUJ1+ RGCs in sham-injured and ONC-injured conditions. Long-term RGC survival 14 days after ONC was not altered by SARM1 deficiency (n ≥ 5 for each genotype; P>0.05 for all comparisons; error bars, SEM).

Figure 3
Figure 3. SARM1 disruption does not prevent JNK activation in RGC somas after axon injury

A. 5 days after ONC, JNK is active (phosphorylated, pJNK) in RGCs. SARM1 deficiency does not prevent activation of JNK after ONC. B. Similarly, the canonical target of JNKs, JUN, accumulates in RGCs 5 days after ONC and SARM1 deficiency does not prevent JUN accumulation. These data suggest, unlike in other systems, SARM1 is not required for JNK signaling. At least 3 sections from 3 different eyes were assessed for each genotype.

Figure 4
Figure 4. SARM1 deficiency protects RGCs from axon degeneration after ONC

A. Representative images of longitudinal optic nerve sections from mice of the indicated genotypes in which RGC axons were labeled with CFP by crossing the Thy-CFP transgene into the strain (CFP+). Histological signs of axon degeneration such as axon beading and fragmentation were evident in WT optic nerves 5 days after ONC. However, fewer morphological signs of axon degeneration were observed in Sarm1−/−.CFP+ and WldS.CFP+ optic nerves 5 days after ONC (n = 3 for each genotype). B. Representative traces of the CAP recorded from WT, Sarm1−/− and WldS optic nerves 5 days after crush. C. SARM1 deficiency significantly reduced RGC axon degeneration 5 days after crush. The CAP amplitude recorded from Sarm1−/− and WldS nerves was significantly higher than WT nerves 5 days after ONC. Interestingly, the CAP amplitude was not significantly different in Sarm1−/− and WldS nerves 5 days after ONC (n ≥ 5 for each genotype and condition; error bars, SEM). Note, Sarm1+/+ and WldS littermate controls that did not carry the mutation were grouped together in the WT group.

Figure 5
Figure 5. SARM1 loss and WldS function in the same axon degeneration pathway

To determine if SARM1 and WLDS function in the same axon degeneration pathway, mice expressing WldS in a Sarm1-null background were generated. Axon degeneration was assessed by quantifying the maximal amplitude of the CAP 14 days after ONC. The data for Sarm1−/− and WldS mice are the same as from Figure 1. The CAP amplitude of all genotypes was significantly reduced 14 days after ONC, however, and Sarm1−/−, WldS, and Sarm1−/−;WldS mice all had significantly higher CAPs compared to WT controls. There was no difference in CAPs from Sarm1−/−;WldS mice compared to WldS or Sarm1−/− mice (n ≥ 5 for each genotype and condition).

Figure 6
Figure 6. DR6 deficiency does not protect RGCs from somal or axonal degeneration after axonal injury

A. Representative images from WT and DR6−/− flat-mounted retinas stained with anti-cleaved caspase 3 (cCASP3) 5 days after ONC. B. Cell counts of the number of cCASP3 positive cells 5 days after crush (n ≥ 5 for each genotype) showed no significant difference between WT and DR6−/− mice (P>0.05). C. Representative traces of the compound action potential (CAP) recorded from WT and DR6−/− optic nerves 5 days after crush. D. The CAP amplitude was significantly reduced in both WT and DR6−/− optic nerves 5 days after crush. However, the reduction of CAP amplitude observed in DR6 deficient mice was similar to that observed in control mice (P>0.05; (n ≥ 5 for each genotype and condition), suggesting DR6 is not an important component of the axonal degeneration pathway in adult RGCs. Scale bar: A, 50 m; error bars, SEM).

Figure 7
Figure 7. Summary diagram of somal and axonal degeneration pathways controlling RGC somal and axonal degeneration after mechanical optic nerve injury

After mechanical optic nerve injury (ONC) distinct injury signaling pathways appear to control somal (proximal to the site of injury) and axonal (distal to the site of injury) degeneration. Proximal to the site of injury a MAPK pathway that includes DLK/LZK and JNK2/3 activation activates the transcription factor JUN. JUN activation, presumably through altering the transcription of injured RGCs, leads to BAX activation and somal apoptosis. Also, an ER stress pathway and other factors have been shown to regulate RGC death after axonal injury. Axonal degeneration distal to the site of axonal injury is less well defined. After ONC injury, SARM1 deficiency lessons axonal degeneration; thus, the presence of SARM1 facilitates axonal degeneration presumably by consuming NAD+. The MAPK pathway, particularly the MAP2Ks (MKK4 and MKK7) and DLK have also been implicated in axonal degeneration. It is unclear (gray arrows) how important these molecules are for axonal degeneration in RGCs and whether they are upstream or downstream of SARM1 dependent events. “?” represents a presumed step(s) in the pathway that involve an unknown molecule(s).

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