Fam134c and Fam134b shape axonal endoplasmic reticulum architecture in vivo

Endoplasmic reticulum (ER) remodeling is vital for cellular organization. ER-phagy, a selective autophagy targeting ER, plays an important role in maintaining ER morphology and function. The FAM134 protein family, including FAM134A, FAM134B, and FAM134C, mediates ER-phagy. While FAM134B mutations are linked to hereditary sensory and autonomic neuropathy in humans, the physiological role of the other FAM134 proteins remains unknown. To address this, we investigate the roles of FAM134 proteins using single and combined knockouts (KOs) in mice. Single KOs in young mice show no major phenotypes; however, combined Fam134b and Fam134c deletion (Fam134b/cdKO), but not the combination including Fam134a deletion, leads to rapid neuromuscular and somatosensory degeneration, resulting in premature death. Fam134b/cdKO mice show rapid loss of motor and sensory axons in the peripheral nervous system. Long axons from Fam134b/cdKO mice exhibit expanded tubular ER with a transverse ladder-like appearance, whereas no obvious abnormalities are present in cortical ER. Our study unveils the critical roles of FAM134C and FAM134B in the formation of tubular ER network in axons of both motor and sensory neurons.

(A) Quantification of the tibial sciatic nerve area shows a significant decrease in Fam134b/c dKO compared with the other genotypes.(B) Representative immunofluorescence staining of ChAT (red) and NF200 (green) in tibial sciatic nerve sections from 4-weeks-old WT, Fam134b KO , Fam134c KO     (A) Heatmap reporting the top 50 statistically significant lipids and comparing WT, Fam134b KO , Fam134c KO , and Fam134b/c dKO .The log2 relative lipid quantity scale is depicted on the top right.(B) Lipid concentration in WT, Fam134b KO , Fam134c KO , and Fam134b/c dKO sciatic nerves grouped in lipid subclasses.Lipid quantity is expressed in pmol and normalized to µg of total protein.n = 5 animals/group, 2 replicates/animal.Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparisons test.Data represent mean ± SEM. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Statistical analysis and exact P values are included in the source data files.Source data are available online for this figure.

Figure EV2 .
Figure EV2.Early onset of axonal neurodegeneration and denervation of neuromuscular junctions in Fam134b/c dKO .
and Fam134b/c dKO mice.Scale bar, 50 µm.(C, D) Number of NF200 + (C) or ChAT + (D) axons per mm 2 showing no difference among genotypes.(E, F) Diameter distribution of NF200 + (E) or ChAT + (F) axons showing accumulation of smaller sized axons in Fam134b/c dKO compared with the other genotypes.(G) Representative immunofluorescence staining of AchR (red), NF200 (gray), and Syn1 (green) in EDL muscle from 4-weeks-old WT, Fam134b KO , Fam134c KO , and Fam134b/c dKO mice.Scale bars, 100 µm and 25 µm, respectively, in the ×20 and ×63 magnification.(H) Fam134b/c dKO muscles show an increased percentage of denervated neuromuscular junctions (NMJs) compared with the other genotypes.(I) AchR mean positive area indicates no main changes among the different genotypes.(J) Fam134b/c dKO NMJs show a decreased number of branches compared with the other genotypes.(K) The ratio between the Syn1 and AChR positive area is reduced in Fam134b/c dKO NMJs compared to the other genotypes.Data information: n ≥ 4 animals/group in all experiments.Statistical significance was determined by one-way ANOVA (A, C, D, H-K) or two-way ANOVA (E, F), followed by Tukey's multiple comparisons test.Data represent mean ± SEM. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.Statistical analysis and exact P values are included in the source data files.Source data are available online for this figure.

Figure EV3 .
Figure EV3.Schwann cell, motoneuron, and sensory neuron soma show no ultrastructural alteration in Fam134b/c dKO mice.(A, B) Quantification of the g-ratio of WT, Fam134b KO , Fam134c KO , and Fam134b/c dKO myelinated axons from tibial nerves aged 4 (A) and 15 (B) weeks.n = 3 animals/group.Data represent mean ± SEM. (C) Representative electron micrographs showing Schwann cells in WT and Fam134b/c dKO sciatic nerve aged 4 weeks.Scale bar, 2 µm.(D) Representative electron micrographs of motoneurons in the ventral horn of the lumbar spinal cord from WT and Fam134b/c dKO mice aged 4 weeks.(E) Representative electron micrographs of neurons in lumbar DRG from WT and Fam134b/c dKO mice aged 4 weeks.Scale bars, 1000 nm or 500 nm in the insets.Source data are available online for this figure.

Figure EV4 .
Figure EV4.Autophagy and histology of sciatic nerve show no major alterations in 2-week-old Fam134b/c dKO mice.(A) Western blot analysis showing protein expression of autophagy markers (Sqstm1/p62 and Lc3b), the ER marker Calnexin, and ER-phagy receptors (Atl3, Tex264, Ccpg1, and Rtn3) in sciatic nerve of 2-week-old mice with the indicated genotypes.(B-D) Protein level quantification of autophagy markers (B), Calnexin (C), and ER-phagy receptors (D).Normalized to vinculin.n ≥ 4 animals/group.(E) Representative immunofluorescence staining of ChAT (red) and NF200 (green) in tibial sciatic nerve sections of WT and Fam134b/c dKO mice aged 2 weeks.Scale bar, 50 µm.(F) Quantification of the tibial sciatic nerve area showing no difference between WT and Fam134b/ c dKO mice.(G-J) Morphometric analysis of axons shows no alterations between WT and Fam134b/c dKO mice either in both the number of NF200 + (G) and ChAT + axons (H) or in their diameter distribution (I, J). n = 3 animals/group.Data information: Statistical significance was determined by one-way ANOVA (B-D) followed by Tukey's multiple comparisons test or Student's t-test (F-H).Data represent mean ± SEM. ns P > 0.05, *P < 0.05, **P < 0.01.Statistical analysis and exact p-values are included in the source data files.Source data are available online for this figure.