Neuronal expression of GalNAc transferase is sufficient to prevent the age-related neurodegenerative phenotype of complex ganglioside-deficient mice

Abstract

Gangliosides are widely expressed sialylated glycosphingolipids with multifunctional properties in different cell types and organs. In the nervous system, they are highly enriched in both glial and neuronal membranes. Mice lacking complex gangliosides attributable to targeted ablation of the B4galnt1 gene that encodes β-1,4-N-acetylegalactosaminyltransferase 1 (GalNAc-transferase; GalNAcT(-/-)) develop normally before exhibiting an age-dependent neurodegenerative phenotype characterized by marked behavioral abnormalities, central and peripheral axonal degeneration, reduced myelin volume, and loss of axo-glial junction integrity. The cell biological substrates underlying this neurodegeneration and the relative contribution of either glial or neuronal gangliosides to the process are unknown. To address this, we generated neuron-specific and glial-specific GalNAcT rescue mice crossed on the global GalNAcT(-/-) background [GalNAcT(-/-)-Tg(neuronal) and GalNAcT(-/-)-Tg(glial)] and analyzed their behavioral, morphological, and electrophysiological phenotype. Complex gangliosides, as assessed by thin-layer chromatography, mass spectrometry, GalNAcT enzyme activity, and anti-ganglioside antibody (AgAb) immunohistology, were restored in both neuronal and glial GalNAcT rescue mice. Behaviorally, GalNAcT(-/-)-Tg(neuronal) retained a normal "wild-type" (WT) phenotype throughout life, whereas GalNAcT(-/-)-Tg(glial) resembled GalNAcT(-/-) mice, exhibiting progressive tremor, weakness, and ataxia with aging. Quantitative electron microscopy demonstrated that GalNAcT(-/-) and GalNAcT(-/-)-Tg(glial) nerves had significantly increased rates of axon degeneration and reduced myelin volume, whereas GalNAcT(-/-)-Tg(neuronal) and WT appeared normal. The increased invasion of the paranode with juxtaparanodal Kv1.1, characteristically seen in GalNAcT(-/-) and attributed to a breakdown of the axo-glial junction, was normalized in GalNAcT(-/-)-Tg(neuronal) but remained present in GalNAcT(-/-)-Tg(glial) mice. These results indicate that neuronal rather than glial gangliosides are critical to the age-related maintenance of nervous system integrity.

Keywords: ganglioside; glycosyltransferase; neurodegeneration; transgenic.

Figure 1.

Expression of GalNAcT and complex gangliosides in rescue mice. A, Constructs were generated to drive GalNAcT expression in the neurons and glia of GalNAcT^−/− mice by promoting the GalNAcT gene under the control of the hNFL and mPLP promoters, respectively. B, Ganglioside biosynthetic pathway. The GalNAcT enzyme is necessary for generation of complex gangliosides (surrounded by the green box). C, TLC of extracts from brains of WT, GalNAcT^−/−, GalNAcT^−/−-Tg(neuronal), and GalNAcT^−/−-Tg(glial) mice, sprayed with resorcinol to label gangliosides. Standards for the complex gangliosides GM1, GD1a, GD1b, and GT1b are labeled in the right lane. WT extracts contain all complex gangliosides, which are absent from GalNAcT^−/− extract and restored in GalNAcT^−/−-Tg(neuronal) and GalNAcT^−/−-Tg(glial) mice. GalNAcT^−/− extracts are enriched with simple gangliosides. GalNAcT^−/−-Tg(neuronal) and GalNAcT^−/−-Tg(glial) mice extracts are also enriched with simple gangliosides and additionally express the complex gangliosides but at lower levels than WT. D, Chromatograms of extracted major gangliosides in mouse brains of all genotypes confirm TLC findings. All major gangliosides can be putatively detected. The complex gangliosides GT1b, GD1b, GD1a, and GM1 are the most abundant gangliosides found in WT extracts. These are also present in rescue mice at lower levels. GD3, 9-O-Ac(etyl)-GD3, and GM3 are highly abundant in GalNAcT^−/− extracts and remain at high levels in both rescue mice.

Figure 2.

Restoration of complex ganglioside expression in PNS and CNS tissue identified by anti-ganglioside mAb immunostaining. Complex ganglioside at the NMJ (PNS) and in the ventral spinal cord (CNS) are labeled by mouse monoclonal AgAb, followed by detection with a fluorescently labeled anti-mouse IgG antibody (green in merged images). In nerve–muscle preparations, the NMJs are identified by fluorescently conjugated BTx, which binds the AChR on the postsynaptic membrane. The axons of ventral columns in spinal cord sections are identified by anti-neurofilament antibody (red). Complex gangliosides are present on nerve fiber axolemma in WT and GalNAcT^−/−-Tg(neuronal) mice in both preparations and absent in the GalNAcT^−/− mice. Perisynaptic Schwann cells at the motor nerve terminal of the GalNAcT^−/−-Tg(glial) mice are positive for AgAb, and AgAb immunoreactivity also surrounds the neurofilament marker in the ventral spinal cord. Scale bars, 10 μm.

Figure 3.

Neuronal but not glial expression of complex gangliosides in GalNAcT^−/− mice rescues behavioral deficiencies. A, Hindpaw extension on lifting by the tail occurs in WT mice; this is replicated in GalNAcT^−/−-Tg(neuronal) mice, but abnormal clasping of the hindpaws occurs in GalNAcT^−/− and GalNAcT^−/−-Tg(glial) mice. Grip strength (B), latency to fall from the rotarod (C), and number of foot slips (D) are comparable among genotypes at 2 months. There is significant deterioration with age in GalNAcT^−/− and GalNAcT^−/−-Tg(glial) mice (2-way ANOVA, p < 0.001) compared with WT and GalNAcT^−/−-Tg(neuronal). *** indicates significance compared with WT; ### signifies significance compared with neuronal rescue. *p < 0.05; *** and ^###, p < 0.001.

Figure 4.

Prevention of morphological abnormalities in neuronal but not glial rescue mice. A, Degenerate axon density or number is greater in 12-month-old GalNAcT^−/− mice and reaches significance in OpN and SN compared with age-matched WT mice. Neuronal expression of complex gangliosides significantly attenuates this degeneration, whereas degenerate axon density and number in glial rescue mice remains significantly greater in spinal cord and SN, respectively. B, Myelin volume is significantly reduced in all tissues studied for GalNAcT^−/− and GalNAcT^−/−-Tg(glial) mice compared with WT and also compared with GalNAcT^−/−-Tg(neuronal) in SN. C, Representative EM and light microscopic images from transverse sections of spinal cord, OpN, and SN for all genotypes show normalization of axon and myelin in neuronal rescue mice and increased degenerate axons (indicated by red arrowheads), myelin thinning, and poorer ultrastructure in GalNAcT^−/− and GalNAcT^−/−-Tg(glial) mice. Organelle-filled axons and redundant myelin occurred frequently in GalNAcT^−/− and GalNAcT^−/−-Tg(glial) mice OpN and are indicated by red asterisks and arrows, respectively. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: Cord, 2 μm; OpN, 1 μm; SN, 25 μm.

Figure 5.

Restoration of normal nodal architecture by expression of complex gangliosides in neurons of GalNAcT^−/− mice. A, Representative illustrative images per genotype of Caspr (green) and Kv1.1 (magenta) immunoreactivity at SN and OpN NoR in 6-month-old mice. In SN Kv1.1, invasion into the PN is indicated by orange arrows (SN and OpN) and Caspr protrusions (SN only) by white arrows. B, Invasion of the PN (identified by Caspr) with JPN marker Kv1.1 staining significantly increased in GalNAcT^−/− and GalNAcT^−/−-Tg(glial) mice compared with WT and GalNAcT^−/−-Tg(neuronal) mice SN. Consequently, the distance between Kv1.1-positive domains significantly decreased for both genotypes. This distance was lengthened in GalNAcT^−/−-Tg(neuronal) mice compared with WT. The number of PN Caspr staining protrusions significantly increased for GalNAcT^−/− and GalNAcT^−/−-Tg(glial) SN compared with WT and GalNAcT^−/−-Tg(neuronal) levels, which were comparable. Compared with WT nerve, Caspr staining was significantly lengthened in GalNAcT^−/−-Tg(neuronal) nerve. Conversely, Caspr staining length was significantly shorter in GalNAcT^−/− and GalNAcT^−/−-Tg(glial) SN compared with GalNAcT^−/−-Tg(neuronal). The length of Nav1.6 immunostaining significantly increased in GalNAcT^−/− and both rescue mice compared with WT. C, To scale, schematic representing the length of staining in each domain per genotype. One-way ANOVA, p < 0.05. * signifies significance compared with WT; # signifies significance compared with GalNAcT^−/−-Tg(neuronal). * and ^#, p < 0.05; ** and ^##, p < 0.01; *** and ^###, p < 0.001. Scale bar, 10 μm.

Figure 6.

Transverse bands are maintained by expression of complex gangliosides in neurons and glia of GalNAcT^−/− mice. Representative longitudinal SN EM images demonstrate normal NoR ultrastructural features in WT mice, including ordered arrangement of PN loops and appropriate formation of transverse bands between the axon and glial membranes (visible in enlarged inset). Transverse bands are often absent in GalNAcT^−/− mice but can be identified in both GalNAcT^−/−-Tg(neuronal) and GalNAcT^−/−-Tg(glial) mice. In GalNAcT^−/− and both rescue mice, PN loops aberrantly stack and do not all make appropriate contact with the axolemma. Scale bars, 0.5 μm.

Figure 7.

Peripheral nerve functional recovery in rescue mice. Analysis of extracellular recordings from 4- to 6-month-old GalNAcT^−/−, GalNAcT^−/−-Tg(neuronal), and Tg(glial) SN show a reduction in CV that does not reach significance (A). B, Rate of rise of the compound nerve action potential is significantly reduced compared with WT for all other genotypes (one-way ANOVA, p < 0.05). C, Representative trace for each genotype from which analysis was performed. D, The amplitude of the second pulse per interstimulus interval in paired pulse recordings were plotted per genotype; there were no significant changes to refractory period (two-way ANOVA, p > 0.05). ** and *** signifies significance compared with WT. **p < 0.01, ***p < 0.001. Individual mice data are plotted to demonstrate the variance in the recordings among animals.