Jason D Hinman

The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

We have previously shown that myelin abnormalities characterize the normal aging process of the brain and that an age-associated reduction in Klotho is conserved across species. Predominantly generated in brain and kidney, Klotho overexpression extends life span, whereas loss of Klotho accelerates the development of aging-like phenotypes. Although the function of Klotho in brain is unknown, loss of Klotho expression leads to cognitive deficits. We found significant effects of Klotho on oligodendrocyte functions, including induced maturation of rat primary oligodendrocytic progenitor cells (OPCs) in vitro and myelination. Phosphoprotein analysis indicated that Klothos downstream effects involve Akt and ERK signal pathways. Klotho increased OPC maturation, and inhibition of Akt or ERK function blocked this effect on OPCs. In vivo studies of Klotho knock-out mice and control littermates revealed that knock-out mice have a significant reduction in major myelin protein and gene expression. By immunohistochemistry, the number of total and mature oligodendrocytes was significantly lower in Klotho knock-out mice. Strikingly, at the ultrastructural level, Klotho knock-out mice exhibited significantly impaired myelination of the optic nerve and corpus callosum. These mice also displayed severe abnormalities at the nodes of Ranvier. To decipher the mechanisms by which Klotho affects oligodendrocytes, we used luciferase pathway reporters to identify the transcription factors involved. Together, these studies provide novel evidence for Klotho as a key player in myelin biology, which may thus be a useful therapeutic target in efforts to protect brain myelin against age-dependent changes and promote repair in multiple sclerosis.

Age-dependent myelin degeneration and proteolysis of oligodendrocyte proteins is associated with the activation of calpain-1 in the rhesus monkey.

Myelin provides important insulating properties to axons allowing for propagation of action potentials over large distances at high velocity. Disruption of the myelin sheath could therefore contribute to cognitive impairment, such as that observed during the normal aging process. In the present study, age‐related changes in myelin, myelin proteins and oligodendrocyte proteins were assessed in relationship to calpain‐1 expression and cognition in the rhesus monkey. Isolation of myelin fractions from brain white matter revealed that as the content of the intact myelin fraction decreased with age, there was a corresponding increase in the floating or degraded myelin fraction, suggesting an increased breakdown of intact myelin with age. Of the myelin proteins examined, only the myelin‐associated glycoprotein decreased with age. Levels of the oligodendrocyte‐specific proteins 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and myelin/oligodendrocyte‐specific protein (MOSP) increased dramatically in white matter homogenates and myelin with age. Age‐related increases in degraded CNPase also were demonstrable in white matter in association with increases in activated calpain‐1. Degraded CNPase was also detectable in myelin fractions, with only the floating fraction containing activated calpain‐1. The increases in the activated enzyme in white matter were much greater than those found in myelin fractions suggesting a source other than the myelin membrane for the marked overexpression of activated calpain‐1 with age. In addition, CNPase was demonstrated to be a substrate for calpain in vitro. In summary, changes in myelin and oligodendrocyte proteins occur with age, and they appear to have a significant relationship to cognitive impairment. The overexpression of CNPase and MOSP suggests new formation of myelin by oligodendrocytes, which may occur in response to myelin degradation and injury caused by proteolytic enzymes such as calpain.

Remodeling of the Axon Initial Segment After Focal Cortical and White Matter Stroke

Background and Purpose— Recovery from stroke requires neuroplasticity within surviving adjacent cortex. The axon initial segment (AIS) is the site of action potential initiation and a focal point for tuning of neuronal excitability. Remodeling of the AIS may be important to neuroplasticity after stroke. Methods— Focal cortical stroke in forelimb motor cortex was induced by photothrombosis and compared with sham controls. White matter stroke was produced through stereotactic injection of a vasoconstrictor together with biotinylated dextran amine to retrogradely label injured cortical neurons. AIS length, morphology and number were measured using immunofluorescence and confocal microscopy 2 weeks after stroke. Results— Within the peri-infarct cortex and after white matter stroke, AIS length decreases. This shortening is accompanied by altered AIS morphology. In peri-infarct cortex, the decrease in AIS length after stroke occurs from the distal end of the AIS, resulting in a Nav1.6. &ggr;-aminobutyric acid type A receptor-&agr;2 subunit staining at axoaxonic synapses along the AIS is significantly decreased. In addition, a significant increase in small, immature initial segments is present in layers 2/3 of peri-infarct cortex, reflecting maturation of axonal sprouting and new initial segments from surviving neurons. Conclusions— Stroke alters the compartmental morphology of surviving adjacent neurons in peri-infarct cortex and in neurons whose distal axons are injured by white matter stroke. With a key role in modulation of neuronal excitability, these changes at the AIS may contribute to altered neuronal excitability after injury and prove crucial to increasing neuroplasticity in surviving tissue affected by stroke.

Age-related molecular reorganization at the node of Ranvier

In myelinated axons, action potential conduction is dependent on the discrete clustering of ion channels at specialized regions of the axon, termed nodes of Ranvier. This organization is controlled, at least in part, by the adherence of myelin sheaths to the axolemma in the adjacent region of the paranode. Age‐related disruption in the integrity of internodal myelin sheaths is well described and includes splitting of myelin sheaths, redundant myelin, and fluctuations in biochemical constituents of myelin. These changes have been proposed to contribute to age‐related cognitive decline; in previous studies of monkeys, myelin changes correlate with cognitive performance. In the present study, we hypothesize that age‐dependent myelin breakdown results in concomitant disruption at sites of axoglial contact, in particular at the paranode, and that this disruption alters the molecular organization in this region. In aged monkey and rat optic nerves, immunolabeling for voltage‐dependent potassium channels of the Shaker family (Kv1.2), normally localizing in the adjacent juxtaparanode, were mislocalized to the paranode. Similarly, immunolabeling for the paranodal marker caspr reveals irregular caspr‐labeled paranodal profiles, suggesting that there may be age‐related changes in paranodal structure. Ultrastructural analysis of paranodal segments from optic nerve of aged monkeys shows that, in a subset of myelinated axons with thick sheaths, some paranodal loops fail to contact the axolemma. Thus, age‐dependent myelin alterations affect axonal protein localization and may be detrimental to maintenance of axonal conduction. J. Comp. Neurol. 495:351–362, 2006.

Visualization of APP dimerization and APP‐Notch2 heterodimerization in living cells using bimolecular fluorescence complementation

We previously demonstrated that the amyloid precursor protein (APP) interacts with Notch receptors. Here, we confirmed the APP/Notch1 endogenous interaction in embryonic day 17 rat brain tissue, suggesting the interaction was not as a result of over‐expression artifacts. To investigate potential homodimeric and heterodimeric interactions of APP and Notch2 (N2), we have visualized the subcellular localization of the APP/N2 complexes formed in living cells using bimolecular fluorescence complementation (BiFC) analysis. BiFC was accomplished by fusing the N‐terminal fragment or the C‐terminal fragment of yellow fluorescent protein (YFP) to APP, N2, and a C‐terminally truncated form of N2. When expressed in COS‐7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP homodimer produced a weak fluorescent signal, while neither full‐length N2, nor a truncated N2 alone, produced a visible signal, suggesting that N2 receptors do not form homodimers. The strongest fluorescent signal was obtained with co‐expression of the C‐terminal fragment of YFP fused to APP and the N‐terminal fragment of YFP fused to the truncated form of N2. This heterodimer localized to plasma membrane, endoplasmic reticulum (ER), Golgi and other compartments. The results were confirmed and quantified by flow cytometry. The BiFC method of specifically visualizing APP/Notch interactions can be applied to study APP and Notch signaling during development, aging and neurodegeneration.

What’s Behind the Decline? The Role of White Matter in Brain Aging

The specific molecular events that underlie the age-related loss of cognitive function are poorly understood. Although not experimentally substantiated, age-dependent neuronal loss has long been considered central to age-related cognitive decline. More recently, age-related changes in brain white matter have taken precedence in explaining the steady decline in cognitive domains seen in non-diseased elderly. Characteristic alterations in the ultrastructure of myelin coupled with evidence of inflammatory processes present in the white matter of several different species suggest that specific molecular events within brain white matter may better explain observed pathological changes and cognitive deficits. This review focuses on recent evidence highlighting the importance of white matter in deciphering the course of “normal” brain aging.

Models That Matter: White Matter Stroke Models

SummaryStroke is a devastating neurological disease with limited functional recovery. Stroke affects all cellular elements of the brain and impacts areas traditionally classified as both gray matter and white matter. In fact, stroke in subcortical white matter regions of the brain accounts for approximately 30% of all stroke subtypes, and white matter injury is a component of most classes of stroke damage. However, most basic scientific information in stroke cell death and neural repair relates principally to neuronal cell death and repair. Despite an emerging biological understanding of white matter development, adult function, and reorganization in inflammatory diseases, such as multiple sclerosis, little is known of the specific molecular and cellular events in white matter ischemia. This limitation stems in part from the difficulty in generating animal models of white matter stroke. This review will discuss recent progress in studies of animal models of white matter stroke, and the emerging principles of cell death and repair in oligodendrocytes, axons, and astrocytes in white matter ischemic injury.

Amyloid precursor protein interacts with notch receptors.

The amyloid precursor protein (APP) must fulfill important roles based on its sequence conservation from fly to human. Although multiple functions for APP have been proposed, the best‐known role for this protein is as the precursor of Aβ peptide, a neurotoxic 39–43‐amino acid peptide crucial to the pathogenesis of Alzheimers disease. To investigate additional roles for APP with an eye toward understanding the molecular basis of the pleiotropic effects ascribed to APP, we isolated proteins that interacted with the plasma membrane isoform of APP. We employed a membrane‐impermeable crosslinker to immobilize proteins binding to transmembrane APP in human embryonic kidney (HEK)293 cells expressing APP751 (HEK275) or rat embryonic day 18 primary neurons infected with a virus expressing APP. Notch2 was identified as a potential APP binding partner based on mass spectrometry analysis of APP complexes immunopurified from neurons. To confirm the interaction between Notch2 and APP, we carried out immunoprecipitation studies in HEK275 cells transiently expressing full‐length Notch2 using Notch2 antibodies. The results indicated that APP and Notch2 interact in mammalian cells, and confirmed our initial findings. Interestingly, Notch1 also coimmunoprecipitated with APP, suggesting that APP and Notch family members may engage in intermolecular cross talk to modulate cell function. Finally, cotransfection of APP/CFP and Notch2/YFP into COS cells revealed that these two proteins colocalize on the plasma membrane. Intracellularly, however, although some APP and Notch molecules colocalize, others reside in distinct locations. The discovery of proteins that interact with APP may aid in the identification of new functions for APP.

Activation of calpain-1 in myelin and microglia in the white matter of the aged rhesus monkey

Ultrastructural disruption of myelin sheaths and a loss of myelin with age are well‐documented phenomena in both the human and rhesus monkey. Age‐dependent activation of calpain‐1 (EC 3.4.22.52) has been suggested as a plausible mechanism for increased proteolysis in the white matter of the rhesus monkey. The present study documents activation of calpain‐1 throughout brain white matter in aged animals, evidenced by immunodetection of the activated enzyme as well as a calpain‐derived spectrin fragment in both tissue section and Triton X‐100‐soluble homogenate of subcortical white matter from the frontal, temporal, and parietal lobes. Separation of myelin fractions from brain stem tissue into intact and floating myelin confirmed previous reports of an age‐related increase in activated calpain‐1 in the floating fraction. Measurements of calpain‐1 activity using a fluorescent substrate revealed an age‐related increase in calpain‐1 proteolytic activity in the floating myelin fraction consistent with immunodetection of the activated enzyme in this fraction. Double‐immunofluorescence demonstrated co‐localization of activated calpain‐1 with human leukocyte antigen‐DR (HLA‐DR), a marker for activated microglia, suggesting that these cells represent the major source of the increase in activated calpain‐1 in the aging brain. These data solidify the role of calpain‐1 in myelin protein metabolism and further implicate activated microglia in the pathology of the aging brain.

Age-dependent accumulation of ubiquitinated 2′,3′-cyclic nucleotide 3′-phosphodiesterase in myelin lipid rafts

Changes in brain white matter are prominent features of the aging brain and include glial cell activation, disruption of myelin membranes with resultant reorganization of the molecular components of the node of Ranvier, and loss of myelinated fibers associated with inflammation and oxidative stress. In previous studies, overexpression of CNP, a key myelin protein, was implicated in age‐related changes in myelin and axons. Here we examine the extent of CNP accumulation in brain white matter and isolated myelin of aged rhesus monkeys and its relationship to CNP degradation and partitioning in myelin. With age, excess CNP is found in myelin and throughout brain white matter accompanied by proteolytic fragments of CNP. These increases occur in the absence of changes in CNP mRNA levels. Using a combination of 2D electrophoresis, immunoprecipitation, and mass spectrometry analysis, ubiquitinated CNP was demonstrable in the Triton X‐100 insoluble lipid raft associated fractions of myelin isolated from rhesus monkeys. Further, using ubiquitin‐mediated fluorescence complementation (UbFC), ubiquitinated CNP was visualized by microscopy in both COS‐7 and MO3.13 cells and by immunoblot in MO3.13 cells and appears to at least partially localize within lipid rafts. The findings suggest that incomplete degradation of CNP due to failure of the proteasomal system and aberrant degradation by calpain‐1 leads to age‐related CNP accumulation and proteolysis. In sum, we suspect these phenomena result in age‐related dysfunction of CNP in the lipid raft, which may lead to myelin and axonal pathology.