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. 2016 Apr 18;10:42.
doi: 10.3389/fnana.2016.00042. eCollection 2016.

HERC 1 Ubiquitin Ligase Mutation Affects Neocortical, CA3 Hippocampal and Spinal Cord Projection Neurons: An Ultrastructural Study

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Free PMC article

HERC 1 Ubiquitin Ligase Mutation Affects Neocortical, CA3 Hippocampal and Spinal Cord Projection Neurons: An Ultrastructural Study

Rocío Ruiz et al. Front Neuroanat. .
Free PMC article

Abstract

The spontaneous mutation tambaleante is caused by the Gly483Glu substitution in the highly conserved N terminal RCC1-like domain of the HERC1 protein, which leads to the increase of mutated protein levels responsible for cerebellar Purkinje cell death by autophagy. Until now, Purkinje cells have been the only central nervous neurons reported as being targeted by the mutation, and their degeneration elicits an ataxic syndrome in adult mutant mice. However, the ultrastructural analysis performed here demonstrates that signs of autophagy, such as autophagosomes, lysosomes, and altered mitochondria, are present in neocortical pyramidal, CA3 hippocampal pyramidal, and spinal cord motor neurons. The main difference is that the reduction in the number of neurons affected in the tambaleante mutation in the neocortex, the hippocampus, and the spinal cord is not so evident as the dramatic loss of cerebellar Purkinje cells. Interestingly, signs of autophagy are absent in both interneurons and neuroglia cells. Affected neurons have in common that they are projection neurons which receive strong and varied synaptic inputs, and possess the highest degree of neuronal activity. Therefore, because the integrity of the ubiquitin-proteasome system is essential for protein degradation and hence, for normal protein turnover, it could be hypothesized that the deleterious effects of the misrouting of these pathways would depend directly on the neuronal activity.

Keywords: autophagy; cerebellum; cerebral cortex; hippocampus; neuron; proteasome; spinal cord; ubiquitin.

Figures

Figure 1
Figure 1
Parasagittal sections through the cerebellar cortex of 4-month-old tbl/tbl mice. 1.5 μm-thick sections illustrate that disappeared Purkinje cells are substituted by glial Golgi-epithelial cells (g, A,B). Remaining degenerating Purkinje cells possess degenerative dark accumulations within the cytoplasm of the soma (A,B, asterisk, arrows) and thick, dark dendritic trees (A, small arrow). Note the swelling of glial processes surrounding Purkinje cells (B, arrowhead). Degenerative signs consisting of lysosomes, electron-dense debris, and autophagosomes with different degrees of evolution (arrows in C–F), are present in the dendrites of the molecular layer and in the Purkinje cells’ cytoplasm (arrows, in C,F). Necrotic debris is also engulfed by glial cell processes (D,E, g). n, nucleus of a Purkinje cell. Pf, parallel fiber. Bars = 20 μm (A,B), 2 μm (C), and 0.5 μm (D–F).
Figure 2
Figure 2
Coronal sections through the spinal cord of 4- (A–E) and 1- (F–I) month-old tbl/tbl mice. In the older mutant mice, abundant dark accumulations are observable within the cytoplasm of the cell soma (arrows in C,D) and the dendrites (arrowheads in D,E) of the motor neurons. In contrast, few of these dark aggregates can be found in the younger animals (arrow in I). Bars = 200 μm (A,F), 100 μm (B,G), and 20 μm (C–E,H,I).
Figure 3
Figure 3
Photomontage of a motor neuron from the spinal cord of a 4-month-old tbl/tbl mouse. Arrows indicate lysosomes with different degrees of evolution distributed throughout the cell soma and dendritic cytoplasm (A–D). Multivesicular bodies (mb), incipient autophagosomes (arrowhead), and empty vacuoles are often observed near the lysosomes (B–D). Note that axosomatic and axodendritic synapses present an unaltered morphology (asterisks in A). Bars = 5 μm (A), 1 μm (B), and 0.5 μm (C,D).
Figure 4
Figure 4
Coronal sections through the CA3 of the hippocampus of 4-month-old tbl/tbl mice. 1.5 μm thick sections show dark degenerative accumulations in the pyramidal cell somata (arrows in B). Condensed dark nuclei are occasionally observed within the pyramidal cell layer (arrowhead in B). Dark accumulations and vacuoles found throughout pyramidal cell cytoplasm have the same ultrastructural features (arrows in C–E) as those found in Purkinje cells and spinal motor neurons. Bars = 100 μm (A), 20 μm (B), 2 μm (C), and 1 μm (D–E).
Figure 5
Figure 5
Coronal sections through the frontal cerebral cortex of 4-month-old tbl/tbl mice. 1.5 μm thick sections illustrate dark degenerative accumulations in both pyramidal cell somata (arrows in B–D) and apical dendrites (arrow in B). Cytoplasmic inclusion with characteristics of autophagosomes (arrowhead in H; e) and lysosomes (arrows in H; f,g) are observed within the pyramidal cell bodies. Bars = 100 μm (A), 20 μm (B–D), 2 μm (E), and 0.5 μm (E–G).
Figure 6
Figure 6
Microphotographs of coronal sections through the frontal cerebral cortex of 4-month-old tbl/tbl mice. Lysosomes located within the cytoplasm of pyramidal cell somata (arrows in A; B–D) and dendrites (arrow in A; e) which receive synapses of normal appearance (arrowhead in E). Some pyramidal cells with lysosomes in their cytoplasm show vacuoles within their nuclei (asterisks in A,D). Bars = 2 μm (A,B) and 1 μm (C–E).
Figure 7
Figure 7
Confocal images of coronal sections through the frontal cortex layers II-III (A–C) and CA3 hippocampus (G–I) of 4-month-old wt mice, and the same neocortical (D–F), and hippocampal region (J–L) of 4-month-old tbl/tbl mice. Beclin-1 expression is most pronounced in tbl/tbl brains (B,K), and as in wt co-express with calbindin (CaBP) inmmunoreactive and non-immunoreactive cell bodies (C,F,I,L). IV, layer IV. Bars = 50 μm.
Figure 8
Figure 8
Confocal images of coronal sections through the frontal cortex layers II-III (A–C) and CA3 hippocampus (G–I) of 4-month-old wt mice, and the same neocortical (D–F), and hippocampal region (J–L) of 4-month-old tbl/tbl mice. Light chain 3 (LC3) co-expression with the neuronal marker NeuN is also most pronounced in tbl/tbl brains (F,L) than in wt similar brain regions (C,I). I, layer I; IV, layer IV. Bar = 50 μm.
Figure 9
Figure 9
Confocal images of coronal sections through the frontal cortex layers II-III (A–C) of 4-month-old wt mice, and the frontal cortex (D–F) of 4-month-old tbl/tbl mice. p62 immunoreactive is stronger in tbl/tbl cortex (E) than in wt cortex (B); and in both wt (C) and tbl/tbl (F) co-express in calbindin (CaBP) immunoreactive neuronal cell bodies. I, layer I. Bars = 50 μm.
Figure 10
Figure 10
Histograms of Beclin-1 (A,B), LC3 (C,D), and p62 (E,F) immunoreactivity densities through the CA3 hippocampus and frontal neocortex of 4-month-old wt mice (black bars), and the same brain regions of 4-month-old tbl/tbl mice (white bars). In the hippocampus the differences of immunoreactivity densities are not statistically significant (asterisks), being p > 0.5 for Beclin-1, p = 0.440 for LC3, and p = 0.397 for p62. In contrast, the differences of immunorectivity densities are statistically significant for the three autophagic cycle markers in the cerebral cortex (double asterisks; p = 0.019 for Beclin-1, p = 0.019 for LC3, and p = 0.017 for p62).

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