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. 2004 Apr 6;101(14):5075-80.
doi: 10.1073/pnas.0401030101. Epub 2004 Mar 29.

Brain-specific deletion of neuropathy target esterase/swisscheese results in neurodegeneration

Katerina Akassoglou  1 , Brian Malester, Jixiang Xu, Lino Tessarollo, Jack Rosenbluth, Moses V Chao
Affiliations

Brain-specific deletion of neuropathy target esterase/swisscheese results in neurodegeneration

Katerina Akassoglou et al. Proc Natl Acad Sci U S A. .

Abstract

Neuropathy target esterase (NTE) is a neuronal membrane protein originally identified for its property to be modified by organo-phosphates (OPs), which in humans cause neuropathy characterized by axonal degeneration. Drosophila mutants for the homolog gene of NTE, swisscheese (sws), indicated a possible involvement of sws in the regulation of axon-glial cell interaction during glial wrapping. However, the role of NTE/sws in mammalian brain pathophysiology remains unknown. To investigate NTE function in vivo, we used the cre/loxP site-specific recombination strategy to generate mice with a specific deletion of NTE in neuronal tissues. Here we show that loss of NTE leads to prominent neuronal pathology in the hippocampus and thalamus and also defects in the cerebellum. Absence of NTE resulted in disruption of the endoplasmic reticulum, vacuolation of nerve cell bodies, and abnormal reticular aggregates. Thus, these results identify a physiological role for NTE in the nervous system and indicate that a loss-of-function mechanism may contribute to neurodegenerative diseases characterized by vacuolation and neuronal loss.

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Figures

Fig. 1.
Fig. 1.
Brain specific inactivation of the NTE gene. (A) Diagram of targeting construct and strategy. Exons are indicated as black boxes. Gray box, 3′ internal probe; box, 3′ external probe; triangles, LoxP sites. (B) Genomic Southern blot from WT (lane 1), Nes-cre:NTEfl/+ (lane 2), and Nes-cre:NTEfl/fl (lane 3). Brain DNA was cleaved with SpeI and hybridized with a 32P-labeled 5′ external probe. (C) NTE is missing from the brains of Nes-cre:NTEfl/fl mice. Immunoblot analysis using an antibody against NTE from brain extracts of the cerebrum (Cr) and cerebellum (Cb) from WT, NTEfl/fl, and Nes-cre:NTEfl/fl mice. Heart (H) and cerebrum (Cr) were used as negative (-) and positive (+) controls, respectively. Immunoblot using an antibody against GAPDH was used to ensure equal loading between samples. (D) Nes-cre:NTEfl/fl mice (1.26 ± 0.72%, n = 5) had ≈90% less NTE activity in the brain (P < 0.01), when compared to WT mice (11.41 ± 2.47%, n = 5). NTEfl/fl mice (10.75 ± 3.06%, n = 5) showed no difference when compared to WT control mice. (E) No statistically significant differences were observed for AChE activity. (F) Weight curve of Nes-cre:NTEfl/fl mice (n = 16), NTEfl/fl (n = 14) and NTE+/+ (n = 9) littermate controls. There is no difference between NTEfl/fl and NTE+/+ mice, whereas Nes-cre:NTEfl/fl weighed less after 5 weeks of age (P < 0.0001).
Fig. 2.
Fig. 2.
Deletion of NTE induces hippocampal vacuolation. (A) Extensive vacuolation in the neuropil of the hippocampus of Nes-cre:NTEfl/fl. (B) Normal hippocampal morphology of NTEfl/fl littermate control. Boxes are enlarged in C and D. (C) Vacuolation in the CA2 neuropil area of Nes-cre:NTEfl/fl. (D) Normal CA2 neuropil of NTEfl/fl mice. (E) Hippocampal neuropil of Nes-cre:NTEfl/fl mice contained swollen dendrites with large vacuoles and abnormal membranous inclusions. (F) Normal appearance of 4.5-month-old NTEfl/fl hippocampal neuropil. (Scale bar, 250 μmin A and B;36 μmin C and D;1 μmin E; and 4.5 μmin F.)
Fig. 3.
Fig. 3.
NTE localizes to the ER and its deletion results in redistribution of Nissl substance in the thalamic neurons. (A and B) Silver staining showing neuronal loss in the thalamus of a Nes-cre:NTEfl/fl mice mouse (3 months) (A) when compared to age-matched NTEfl/fl mouse (C). (E, F, and G) Fluorescent Nissl stain showing vacuolated neurons in Nes-cre:NTEfl/fl mice. (D) Normal distribution of Nissl substance in the cell body of NTEfl/fl mice. (E) Six-week-old Nes-cre:NTEfl/fl mouse at the onset of neuronal pathology showing two normal and one vacuolated neuron. (H) Subcellular fractionation of hippocampal neurons shows the presence of NTE in the ribophorin, ER-containing fraction, and not in the GM130, Golgi-containing fraction. +, positive control, cerebrum lysate; -, negative control, heart lysate; I, interface between gradients; T, top of gradient; B, bottom of gradient. Double immunofluorescence for calnexin (J) and NTE (I) in primary hippocampal neurons shows association of NTE with the ER (K). (Scale bar, 36 μmin A-C;25 μmin B-D; and 13 μmin E-G.)
Fig. 4.
Fig. 4.
Intracytoplasmic vacuolation, reticular aggregates and ER abnormalities. (A) A neuronal cell body from a 4.5-month-old Nes-cre:NTEfl/fl with normal nucleus (n) surrounded by cytoplasm (c), which in multiple areas appears swollen and clear or has been replaced by vacuoles (v) extending from the nucleus to the plasma membrane. (B) Neuron from a 4.5-month-old NTEfl/fl control mouse showing granular ER, many free ribosomes, mitochondria, and Golgi apparatus filling the cytoplasm (c) between the nucleolus (n) and the plasma membrane. Surrounding neuropil contains normal axonal and dendritic processes. Vacuolated cell body (C) and dendritic process (D) in Nes-cre:NTEfl/fl showing flattened and sharply angulated membranes. (E) A vacuolated process in the neuropil of Nes-cre:NTEfl/fl mouse filled with abnormal aggregates and smooth membranes. (F) Aggregates display a branching reticular appearance and form tubular networks and angular rods within the dendrites. (G) Pentalaminar structures (arrows) form the branched network in the dendrites of Nes-cre:NTEfl/fl mice. (H) Large abnormal process within the neuropil of Nes-cre:NTEfl/fl mice containing reticular and vacuolar inclusions with multilayered membranes. (I) Normal myelin surrounding nerve fibers at the alveus of the hippocampus of Nes-cre:NTEfl/fl mouse. In some cases, myelin sheaths enclose a clear vacuole which displaces the axoplasm to the periphery (arrows). (Scale bar, 1.4 μm in A; 1 μm in B-D and H; 0.4 μm in E; 0.2 μm in F; 0.1 μm in G; and 1.5 μm in I.)
Fig. 5.
Fig. 5.
Loss of Purkinje cells and behavioral deficits caused by brain deletion of NTE. (A) Normal Purkinje cell appearance and Purkinje dendritic tree extension revealed by silver staining in the molecular layer of the cerebellum of NTEfl/fl mice. (B) Loss of Purkinje trees and reduced dendritic tree extension and complexity by silver stain of the cerebellum of Nes-cre:NTEfl/fl mice. (C) Normal number of calbindin positive Purkinje cells in NTEfl/fl mice. (D) Loss of calbindin positive Purkinje cells from Nes-cre:NTEfl/fl.(E) Normal appearance of MAP-2 positive Purkinje cell dendrites and neurons in the molecular layer of NTEfl/fl mice. (F) Loss of MAP-2 positive dendritic trees in the cerebellum. (G) No behavioral differences observed at a rotarod behavioral test measuring motor coordination between 4-week-old NTEfl/fl (n = 4) and Nes-cre:NTEfl/fl mice (n = 5), P > 0.05. (H) Evident coordination defects of 5-month-old Nes-cre:NTEfl/fl mice (n = 4), when compared to NTEfl/fl littermate controls (n = 7), P < 0.0001. GL, granular layer. (Scale bar, 50 μm.)
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