Background and Purpose-Cellular origin of apolipoprotein E (ApoE) in the human brain and its roles in physiological and pathological conditions remain to be clarified. Methods-Immunolocalization of ApoE was investigated in a series of autopsied human brains with or without infarction.ApoE expression was also estimated on immunoblot on protein extracts from autopsied brains and a cultured neuroblastoma cell line of human origin (GOTO) subjected to an oxidative stress induced by exposure to hydrogen peroxide (0.2 mmol/L). Results-In addition to astrocytes and microglia, neurons and degenerated axons in and around the ischemic foci contained ApoE-like immunoreactivity, which was more intense in recent ischemic foci. Immunoblot demonstrated an increase in expression of ApoE in brain extracts from ischemic lesion, and this increase was also pronounced in the cultured neuroblastoma cell line after the stress. Conclusions-Accumulation of ApoE in neurons in and around ischemic foci of the human brain is related to an increase in ApoE synthesis in neurons, as seen in cultured neuronal cells after oxidative stress. Intrinsic regenerative activity of neuron in reaction to external insults may be related to this increase in ApoE of neuronal origin.

Aquaporin 4 (AQP4), one of the water channel proteins on the plasma membrane of astrocytes, is up-regulated in various conditions with brain edema. Possible participation of AQP4 in various inflammatory lesions, more or less associated with edema, was examined in human autopsied brains. Immunohistochemistry was used to investigate AQP4 expression in autopsied brains with multiple sclerosis (MS), human immunodeficiency virus encephalitis (HIVE) or progressive multifocal leukoencephalopathy (PML). The cellular localization of AQP4 and its relation to inflammatory lesions were then examined with double-labeling immunohistochemistry. AQP4 immunoreactivity (IR) was restricted to astrocytes and localized to their entire processes, including their endfeet facing the abluminal surface of capillaries. In MS brains, AQP4-positive astrocytes were more abundant at the periphery of plaques than in their center, as seen in ischemic foci. Quantification of fluorescent signal demonstrated that AQP4 IR was greatly increased around plaques relative to that in unaffected area. Although the white matter was severely involved in HIVE and PML, AQP4-positive astrocytes were rare in the white matter even around perivascular active inflammatory foci. They were abundant in the gray matter and most prominent in the boundary between the gray and white matter, without apparent relation to inflammatory foci. Some bizarre astrocytes in PML exhibited AQP4 IR. Up-regulation of AQP4 was consistently found in astrocytes in various inflammatory lesions. However, the distribution of AQP4-positve astrocytes differed markedly according to disease and was not necessarily related to brain edema, indicating that functions and regulation of AQP4 in human brains are multiple.

Abstract:al-Syntrophin, a member of dystrophin-associated proteins, is expressed at the sarcolemma and at perivascular astrocytes, and participates in protein-protein interactions through its PDZ domain. Aquaporin-4 (AQP4) is the predominant water channel protein in the brain, and also expressed at the sarcolemma of fast-twitch muscle fibers. AQP4 is concentrated in orthogonal array particles (OAPs), and its expression has been reported to be decreased at the sarcolemma of dystrophin-deficient mdx mice. We examined whether al-syntrophin targets AQP4 at the sarcolemma. Immunohistochemistry showed that AQP4 is absent at the sarcolemma in al-syntrophin knockout mice and that its expression is also lost from the perivascular astrocyte endfeet. On the other hand, expression of AQP4 is not decreased at the sarcolemma of the knockout mice in the neonatal stage. Moreover, AQP4 is expressed in lung, stomach, and kidney of wild-type and al-syntrophin null mice. Our results show that al-syntrophin is a key molecule to localize AQP4 to the sarcolemma of mature fast myofibers and astrocyte endfeet, but AQP4 is targeted to the plasma membrane by different molecules in lung, stomach, and kidney.

A series of human brains with cerebral infarction obtained at autopsy were investigated to clarify the possible contribution of aquaporin 4 (AQP4) to the development of brain edema. Cellular localization of AQP4 and its relation to ischemic foci were examined with double-labeling immunohistochemistry. AQP4 immunoreactivity (IR) was more intense at the periphery of ischemic foci than at their center. Double-labeling study demonstrated that AQP4 IR was restricted to astrocytes and was localized to their entire processes, including their end feet facing the outer surface of capillaries. Moreover, AQP4 IR, detectable in the subpial and subependymal zone in the normal condition, was more intense in the vicinity of ischemic foci. Accumulation of AQP4 IR may reflect its participation in the development of brain edema in human brains by playing a role in the transport of water not only through blood vessel walls but also through pial and ependymal surface of the brain.

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The reduction in AQP4 in DMD muscles results from decreased levels of AQP4 mRNA in DMD myofibers.

Freeze-fracture electron microscopy enabled us to observe the molecular architecture of the biological membranes. We were studying the myofiber plasma membranes of health and disease by using this technique and were interested in the special assembly called orthogonal arrays (OAs). OAs were present in normal myofiber plasma membranes and were especially numerous in fast twitch type 2 myofibers; while OAs were lost from sarcolemmal plasma membranes of severely affected muscles with dystrophinopathy and dysferlinopathy but not with caveolinopathy. In the mid nineties of the last century, the OAs turned out to be a water channel named aquaporin 4 (AQP4). Since this discovery, several groups of investigators have been studying AQP4 expression in diseased muscles. This review summarizes the papers which describe the expression of OAs, AQP4, and other AQPs at the sarcolemma of healthy and diseased muscle and discusses the possible role of AQPs, especially that of AQP4, in normal and pathological skeletal muscles.

We examined whether AQP7 molecules are expressed in the normal skeletal muscle at mRNA and protein levels. Gel electrophoresis of the reverse transcription-polymerase chain reaction (RT-PCR) product of total RNA samples of normal human or mouse muscles by using oligonucleotide primers for human or mouse AQP7 showed a band of 328 or 369 basepairs, which corresponded to the basepair length between two primers of AQP7. The nucleotide sequence of these RT-PCR products coincided with those of human and mouse AQP7. Immunoblot, immunohistochemical and immunoelectron-microscopic studies of the protein were done by using the rabbit antibody against the synthetic peptide of the N-terminal cytoplasmic domain of the human AQP7 molecule. Immunoblot analysis showed that the rabbit antibody against human AQP7 reacted with a protein of approximately 30 kDa molecular weight in extracts of normal human and mouse skeletal muscles, and normal mouse liver. Immunohistochemistry with our anti-AQP7 antibody showed an immunoreaction at the myofiber surface of type 1 and type 2 fibers in human muscles and of type 2 fibers in mouse muscles.