Fig 2: Schematic representation of the molecular mechanisms.(A) The molecular seesaw model on the left shows the WT balance between glutamylase (Ttll1) and deglutamylase (Nna1) on the seesaw’s left side and between glycylase protein (Ttll3) and a presumed deglycylase or other molecular mechanism on the seesaw’s right side. The molecular seesaw model on the right shows the pcd abnormal unbalance between the increased glycylase (measured in response to the net increase in tubulin that contains polyglutamate) and the decreased glycylation contrasting with the tubulin hyperglutamylation in pcd PNs, beginning at about P20. (B) The pathological pathway through which the Nna1 mutation leads to a dark type of P20 pcd PN death. Ttll1, tubulin tyrosine ligase–like 1; Nna1, neuronal nuclear protein induced by axotomy; PNs, Purkinje neurons; pcd, Purkinje cell degeneration.

Fig 3: ER stress and protein synthesis inhibition.(A) Images of Nissl-stained cerebellar cortices show cytoplasmic basal polyribosome mass (inside red meniscus in the right panel) in P15 pcd. Scale bar: 15 µm. (B) Nna1 shRNA induced abnormal polyribosomes (yellow), based on superimposed Nissl staining (green) and Calb (red) in cultured PNs. Scale bar: 15 µm. (C) Major changes of several mRNAs in P20 pcd PNs. (D) Increased ER stress markers (Calr3, Atf6, and Bcl2L11, an apoptotic initiator), and decreased protein synthesis initiator (Elf2ß2) in P20 pcd PNs. (E) Cerebellar cortex images confirm the large increase of Calr3 (green) in P20 pcd PNs. Calb (red) PNs, DAPI (blue) cell nuclei. Scale bar: 15 µm. (F) Several markers show increased Calr3, Atf6, and Bcl2L11, plus decreased Elf2ß2 in PN cultures given Nna1 shRNA (decreased deglutamylase), Ttll1 cDNA, or combined Nna1 shRNA plus Ttll1 cDNA (increased glutamylase). Dot pots with mean ± SD, n = 6; 1-way ANOVA, experimental (Nna1 shDNA, Ttll1 cDNA, and Nna1 shRNA plus Ttll1 cDNA) groups compared with control group, **P < 0.01; no statistical difference among experimental groups or among control, Lv control, and shRNA control groups. (G) Protein synthesis curves show concentration-dependent decreases in PN cultures given Nna1 shRNA, Ttll1 cDNA, or Ttll3 shRNA (decreased glycylase shown as percentage of controls). There were no significant differences among Nna1 shRNA, Ttll1 cDNA, and Ttll3 shRNA treatments. pcd, Purkinje cell degeneration; Nna1, neuronal nuclear protein induced by axotomy; PNs, Purkinje neurons; Calr3, calreticulin 3; Aft6, activating transcription factor 6; Bcl2L11, Elf2ß2, eukaryotic initiator 2ß2; B cell lymphoma 2–like protein 11; Ttll1, tubulin tyrosine ligase–like 1.

Fig 4: Characterization of Nna1, Ttll1, and Ttll3.(A) Nna1 mRNA lacks exons 7–11 in young adult pcdSid cerebrum and cerebellum. (B) Nna1 protein is deficient in young adult pcd cerebellum, hippocampus, and substantia nigra. Dot pots with mean ± SD, n = 4–6; 2-tailed Student’s t test, compared with WT, **P < 0.01. (C) Ttll1 and Ttll3 mRNA were much higher in P20 WT PNs than in WT GNs. The Nna1 mutation reduced Ttll3 mRNA in P20 pcd. Dot pots with mean ± SD, n = 3; 2-tailed Student’s t test, compared with WT, **P < 0.01. (D) Ttll1 and Ttll3 blots were from 2 different gels. Ttll3 protein was decreased in P20 pcd cerebellum. Dot pots with mean ± SD, n = 4–6; 2-tailed Student’s t test, compared with WT, **P < 0.01. (E) Ttll1 protein (green) was equivalent in P20 WT and pcd cerebellum. Calbindin (Calb, red) in PNs, DAPI (blue) in cell nuclei. Scale bar: 50 µm. (F) Ttll3 protein (green) was decreased in P20 pcd PNs, like Calb (red) in the distal area of P20 pcd PN dendritic trees. Scale bar: 30 µm. Nna1, neuronal nuclear protein induced by axotomy; Ttll1, tubulin tyrosine ligase–like 1; pcd, Purkinje cell degeneration; PNs, Purkinje neurons; GNs, granule cell neurons.