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Original Investigation |

Aquaporin 4 Expression and Tissue Susceptibility to Neuromyelitis Optica

Marcelo Matiello, MD1; Janet Schaefer-Klein, BSc1; David Sun, PhD1; Brian G. Weinshenker, MD, FRCPC1
[+] Author Affiliations
1Department of Neurology, Mayo Clinic, Rochester, Minnesota
JAMA Neurol. 2013;70(9):1118-1125. doi:10.1001/jamaneurol.2013.3124.
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Published online

Importance  To understand the predilection for optic nerve and spinal cord pathologic changes in neuromyelitis optica (NMO).

Objective  To evaluate tissue-specific expression (RNA and protein) and supramolecular aggregation of aquaporin 4 (AQP4) in mouse, rat, and human tissues.

Design  Quantitative polymerase chain reaction, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and blue native polyacrylamide gel electrophoresis.

Setting  Laboratory analysis of mouse, rat, and human tissue.

Participants  Tissues from control individuals and 1 patient with NMO obtained at autopsy.

Exposure  Neuromyelitis optica in the patient.

Main Outcomes and Measures  Tissue-specific messenger RNA and protein expression and supramolecular aggregation of AQP4.

Results  The AQP4 messenger RNA and proteins were much more highly expressed in the optic nerve and spinal cord relative to other regions of the brain and to non–central nervous system tissues in all species evaluated. Large supramolecular aggregates of AQP4 were overrepresented in the optic nerve and spinal cord relative to other central nervous system tissue. There was no difference in AQP4 expression between an individual with NMO and the control samples.

Conclusions and Relevance  Optic nerve tissue from an individual with NMO did not differ in AQP4 expression from control samples. The relatively high levels of expression and of supramolecular aggregation in the optic nerve and spinal cord may contribute to the predilection of these structures to NMO pathologic changes.

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Figure 1.
Aquaporin 4 (AQP4) Messenger RNA (mRNA) Expression

Quantitative polymerase chain reaction analysis of AQP4 mRNA expression in 3 BL6 mice using SYBR-Green–based (Invitrogen) chemical analysis (A) and 2 human control tissue samples using SYBR-Green–based and gene expression assay (TaqMan; ABI) chemical analysis (B). Tissues analyzed were liver (L), kidney (K), muscle (M), frontal lobe (Br), brainstem (BS), hypothalamus (HyT), spinal cord (SC), and optic nerve (ON). Results are expressed as a mean ratio of the samples tested relative to a reference gene, hypoxanthine guanine phosphoribosyl transferase 1 (HPRT). In mouse tissue, M1 and M23 AQP4 isoforms were evaluated separately; in human tissue, total AQP4 (M1 and M23 isoforms) mRNA was evaluated. SYBR-Green–based and TaqMan chemical analysis methods show quantitatively different but similar relative levels of expression.

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Figure 2.
Aquaporin 4 (AQP4) Protein Expression

Analysis of AQP4 protein expression by sodium dodecyl sulfate–18% polyacrylamide gel electrophoresis in neuromyelitis optica–relevant tissues from representative mouse (A), rat (B), and human (C) tissues. Proteins analyzed are from the cerebral hemisphere (Br), brainstem (BS), optic nerve (ON), spinal cord (SC), kidney (K), and liver (L). The M1 and M23 lanes were loaded with proteins isolated from HEK293 cells transfected separately with human M1 or M23 AQP4 isoform pcDNA. Loading was optimized to distinguish M1 from M23; differences in β-actin reflect the differences in protein loading. Densitometry results are summarized in the graphs to the right of the gels.

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Figure 3.
Aquaporin 4 (AQP4) Protein Aggregation in Central Nervous System (CNS) Tissue

Analysis of protein aggregation by blue native polyacrylamide gel electrophoresis in CNS tissue from a BL6 mouse (A), Lewis rat (B), and human control (C). Gels were loaded to optimize the visualization of upper-order aggregates. Proteins analyzed are from the cerebral hemisphere (Br), spinal cord (SC), and optic nerve (ON). Protein molecular weights correspond to a protein standard designed for molecular weight estimation of aggregated proteins (NativeMark Protein Standard; Invitrogen).

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Figure 4.
Cerebral Hemisphere (Br) vs Optic Nerve (ON) Aquaporin 4 (AQP4) Aggregation

A, Blue native polyacrylamide gel electrophoresis (BN-PAGE) comparing aggregated AQP4 in ON vs Br tissue. Protein molecular weights correspond to a protein standard designed for molecular weight estimation of aggregated proteins (NativeMark Protein Standard; Invitrogen). B, Two-dimensional protein analysis wherein lanes from a 3% to 12% nondenaturing BN-PAGE gel are cut and electrophoresed in a second dimension on a denaturing sodium dodecyl sulfate–12% polyacrylamide gel (12% SDS-PAGE). The long horizontal arrow is in the direction of electrophoresis for the single-dimension 3% to 12% BN-PAGE. The vertical arrow is in the direction of electrophoresis for the second-dimension 12% SDS-PAGE. The short horizontal arrows indicate the positions of M1 and M23 isoforms of AQP4 protein.

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Figure 5.
Messenger RNA (mRNA), Protein Expression, and Aggregation in a Patient With Neuromyelitis Optica (NMO) Compared With Controls

Comparison of aquaporin 4 (AQP4) mRNA (A), protein (B), and protein aggregation (single-dimension blue native polyacrylamide gel electrophoresis [BN-PAGE] [C] and 2-dimensional BN-PAGE [D]) between a single patient with NMO and human control samples. The optic nerve (ON) was studied using the SYBR-Green (Invitrogen) technique for mRNA expression from a single patient with NMO and from 5 control ONs (A) and from a single control (B through D) in cerebral hemisphere (Br), spinal cord (SC), ON, and kidney (K) for protein expression and aggregation. Messenger RNA expression is intermediate in NMO. Sample loading was performed to optimize the distinction of M1 from M23; β-actin serves as a comparator protein to control for differences in protein loading. Protein molecular weights (C) correspond to a protein standard designed for molecular weight estimation of aggregated proteins (NativeMark Protein Standard; Invitrogen). 12% SDS-PAGE indicates sodium dodecyl sulfate–12% polyacrylamide gel. Arrows are explained in the legend to Figure 4.

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