Ahdeah Pajoohesh-Ganji

The effects of amyloid precursor protein on postsynaptic composition and activity.

The amyloid precursor protein (APP) is cleaved to produce the Alzheimer disease-associated peptide Aβ, but the normal functions of uncleaved APP in the brain are unknown. We found that APP was present in the postsynaptic density of central excitatory synapses and coimmunoprecipitated with N-methyl-d-aspartate receptors (NMDARs). The presence of APP in the postsynaptic density was supported by the observation that NMDARs regulated trafficking and processing of APP; overexpression of the NR1 subunit increased surface levels of APP, whereas activation of NMDARs decreased surface APP and promoted production of Aβ. We transfected APP or APP RNA interference into primary neurons and used electrophysiological techniques to explore the effects of APP on postsynaptic function. Reduction of APP decreased (and overexpression of APP increased) NMDAR whole cell current density and peak amplitude of spontaneous miniature excitatory postsynaptic currents. The increase in NMDAR current by APP was due to specific recruitment of additional NR2B-containing receptors. Consistent with these findings, immunohistochemical experiments demonstrated that APP increased the surface levels and decreased internalization of NR2B subunits. These results demonstrate a novel physiological role of postsynaptic APP in enhancing NMDAR function.

The effects of ABCA1 on cholesterol efflux and Aβ levels in vitro and in vivo

ABCA1 promotes cholesterol efflux from cells and is required for maintaining plasma cholesterol levels. Cholesterol homeostasis is important in the production of β‐amyloid (Aβ), a peptide that is overproduced in Alzheimers disease (AD). Overexpression of ABCA1 can be achieved by stimulating Liver X Receptors (LXR), and changes in Aβ have been reported after LXR stimulation in vitro. To determine whether ABCA1 could alter endogenous Aβ levels, we used two different in vivo systems. We first examined the effects of an LXR agonist (TO‐901317) on wild‐type mice and found an increase in brain ABCA1 and apoE levels, which caused an increase in plasma cholesterol. This was accompanied by a decrease in brain Aβ levels. We then examined endogenous Aβ levels in ABCA1 knockout mice and found that, despite having no ABCA1, lowered brain apoE levels, and lowered plasma cholesterol, there was no change in Aβ levels. To assess these in vivo models in an in vitro system, we designed a model in which cholesterol transport via ABCA1 (or related transporters) was prevented. Switching off cholesterol efflux, even in the presence of TO‐901317, caused no change in Aβ levels. However, when efflux capability was restored, TO‐901317 reduced Aβ levels. These data show that promoting cholesterol efflux is a viable target for Aβ reducing strategies; however, knockout of cholesterol transporters is not sufficient to alter Aβin vitro or in vivo.

In search of markers for the stem cells of the corneal epithelium

The anterior one‐fifth of the human eye is called the cornea. It consists of several specialized cell types that work together to give the cornea its unique optical properties. As a result of its smooth surface and clarity, light entering the cornea focuses on the neural retina allowing images to come into focus in the optical centres of the brain. When the cornea is not smooth or clear, vision is impaired. The surface of the cornea consists of a stratified squamous epithelium that must be continuously renewed. The cells that make up this outer covering come from an adult stem cell population located at the corneal periphery at a site called the corneal limbus. While engaging in the search for surface markers for corneal epithelial stem cells, vision scientists have obtained a better understanding of the healthy ocular surface. In this review, we summarize the current state of knowledge of the ocular surface and its adult stem cells, and analyse data as they now exist regarding putative corneal epithelial stem cell markers.

Integrins in slow-cycling corneal epithelial cells at the limbus in the mouse.

Adult corneal epithelial stem cells (CESCs) have been shown to reside at the periphery of the cornea at a site called the corneoscleral junction or limbus. Although studies have shown that these cells are slow cycling, their molecular characteristics are not well understood. Using a whole‐mount procedure, we show that whereas α9‐integrin is present in a subset of the basal cells at the corneal limbus and absent in the central cornea, β1‐, β4‐, α3‐, and α6‐integrins are more highly expressed overall in central corneal basal cells. To characterize CESCs based on their slow‐cycling nature, we simultaneously evaluated 5‐bromo‐2‐deoxyuridine (BrdU) label‐retaining cells (LRCs) and integrin expression (α9, β1, and β4) in a total of 1,889 cells at the limbus of adult mice that had been injected as neonates with BrdU. Whereas the LRCs were usually observed adjacent to α9‐integrin‐positive cells, most LRCs were α9‐integrin–negative and expressed high levels of β1‐ and β4‐integrin. In addition, we observed more BrdU‐positive LRCs at the superior and inferior quadrants of adult mouse corneas than at the nasal and temporal quadrants, and determined that 0.94 to 3.6% of the limbal basal cells were slow cycling. We conclude from these data that the slow‐cycling LRCs in the adult mouse cornea are enriched in cells that express high levels of β1‐ and β4‐integrin and little α9‐integrin.

Activation of metabotropic glutamate receptor 5 improves recovery after spinal cord injury in rodents.

Activation of metabotropic glutamate receptor 5 (mGluR5) has neuroprotective properties in vitro and has been reported to limit postischemic lesion volume in vivo. Previously, mGluR5 has been identified on microglia in vitro, but the effects of mGluR5 activation on inflammation in vivo or on recovery after spinal cord injury is unknown.

Wounding the cornea to learn how it heals

Corneal wound healing studies have a long history and rich literature that describes the data obtained over the past 70 years using many different species of animals and methods of injury. These studies have lead to reduced suffering and provided clues to treatments that are now helping patients live more productive lives. In spite of the progress made, further research is required since blindness and reduced quality of life due to corneal scarring still happens. The purpose of this review is to summarize what is known about different types of wound and animal models used to study corneal wound healing. The subject of corneal wound healing is broad and includes chemical and mechanical wound models. This review focuses on mechanical injury models involving debridement and keratectomy wounds to reflect the authors expertise.

Cortical Injury Increases Cholesterol 24S Hydroxylase (Cyp46) Levels in the Rat Brain

In traumatic brain injury (TBI), cellular loss from initial impact as well as secondary neurodegeneration leads to increased cholesterol and lipid debris at the site of injury. Cholesterol accumulation in the periphery can trigger inflammatory mechanisms while cholesterol clearance may be anti-inflammatory. Here we investigated whether TBI altered the regulation of cholesterol 24S-hydroxylase (Cyp46), an enzyme that converts cholesterol to the more hydrophilic 24S-hydroxycholesterol. We examined by Western blot and immunohistochemistry changes in Cyp46 expression following fluid percussion injury. Under normal conditions, most Cyp46 was present in neurons, with very little measurable in glia. Cyp46 levels were significantly increased at 7 days post-injury, and cell type specific analysis at 3 days post-injury showed a significant increase in levels of Cyp46 (84%) in microglia. Since 24-hydroxycholesterol induces activation of genes through the liver X receptor (LXR), we examined protein levels of ATP-binding cassette transporter A1 and apolipoprotein E, two LXR regulated cholesterol homeostasis proteins. Apolipoprotein E and ATP-binding cassette transporter A1 were increased at 7 days post-injury, indicating that increased LXR activity coincided with increased Cyp46 levels. We found that activation of primary rat microglia by LPS in vitro caused increased Cyp46 levels. These data suggest that increased microglial Cyp46 activity is part of a system for removal of damaged cell membranes post-injury, by conversion of cholesterol to 24-hydroxycholesterol and by activation of LXR-regulated gene transcription.

A combined scoring method to assess behavioral recovery after mouse spinal cord injury.

Although the rat has been the predominant rodent used to investigate the pathophysiology and treatment of experimental spinal cord injury (SCI), the increasing availability of transgenic animals has led to greater use of mouse models. However, behavioral assessment after SCI in mice has been less extensively investigated than in rats and few studies have critically examined the correlation between behavioral tests and injury severity or tissue damage. The present study characterized hindlimb functional performance in C57Bl/6 mice after contusion SCI at T9 using the weight drop method. A number of behavioral tests were examined with regard to variability, inter-rater reliability, and correlation to injury severity and white matter sparing. Mice were subjected to sham, mild-moderate or moderate-severe SCI and evaluated at day 1 and weekly up to 42 days using the Basso mouse scale (BMS), ladder climb, grid walk, inclined plane, plantar test and tail flick tests. The ladder climb and grid walk tests proved sub-optimal for use in mice, but modifications enhanced their predictive value with regard to injury severity. The inclined plane, plantar test and tail flick test showed far too much variability to have meaningful predictive value. The BMS score proved reliable, as previously reported, but a combined score (BLG) using BMS, Ladder climb (modified), and Grip walk (modified grid walk) provided better separation across injury levels and less variability than the individual tests. These data provide support for use of a combined scoring method to follow motor recovery in mice after contusion SCI.

GGF2 (Nrg1-β3) treatment enhances NG2+ cell response and improves functional recovery after spinal cord injury

The adult spinal cord contains a pool of endogenous glial precursor cells, which spontaneously respond to spinal cord injury (SCI) with increased proliferation. These include oligodendrocyte precursor cells that express the NG2 proteoglycan and can differentiate into mature oligodendrocytes. Thus, a potential approach for SCI treatment is to enhance the proliferation and differentiation of these cells to yield more functional mature glia and improve remyelination of surviving axons. We previously reported that soluble glial growth factor 2 (GGF2)‐ and basic fibroblast growth factor 2 (FGF2)‐stimulated growth of NG2+ cells purified from injured spinal cord in primary culture. This study examines the effects of systemic administration of GGF2 and/or FGF2 after standardized contusive SCI in vivo in both rat and mouse models. In Sprague‐Dawley rats, 1 week of GGF2 administration, beginning 24 h after injury, enhanced NG2+ cell proliferation, oligodendrogenesis, chronic white matter at the injury epicenter, and recovery of hind limb function. In 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterase‐enhanced green fluorescent protein mice, GGF2 treatment resulted in increased oligodendrogenesis and improved functional recovery, as well as elevated expression of the stem cell transcription factor Sox2 by oligodendrocyte lineage cells. Although oligodendrocyte number was increased chronically after SCI in GGF2‐treated mice, no evidence of increased white matter was detected. However, GGF2 treatment significantly increased levels of P0 protein‐containing peripheral myelin, produced by Schwann cells that infiltrate the injured spinal cord. Our results suggest that GGF2 may have therapeutic potential for SCI by enhancing endogenous recovery processes in a clinically relevant time frame.

Delayed cell cycle pathway modulation facilitates recovery after spinal cord injury

Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction through mechanical damage and secondary biochemical and physiological responses. We have previously described the pathobiological role of cell cycle pathways following rat contusion SCI by examining the effects of early intrathecal cell cycle inhibitor treatment initiation or gene knockout on secondary injury. Here, we delineate changes in cell cycle pathway activation following SCI and examine the effects of delayed (24 h) systemic administration of flavopiridol, an inhibitor of major cyclin-dependent kinases (CDKs), on functional recovery and histopathology in a rat SCI contusion model. Immunoblot analysis demonstrated a marked upregulation of cell cycle-related proteins, including pRb, cyclin D1, CDK4, E2F1 and PCNA, at various time points following SCI, along with downregulation of the endogenous CDK inhibitor p27. Treatment with flavopiridol reduced induction of cell cycle proteins and increased p27 expression in the injured spinal cord. Functional recovery was significantly improved after SCI from day 7 through day 28. Treatment significantly reduced lesion volume and the number of Iba-1+ microglia in the preserved tissue and increased the myelinated area of spared white matter as well as the number of CC1+ oligodendrocytes. Furthermore, flavopiridol attenuated expression of Iba-1 and glactin-3, associated with microglial activation and astrocytic reactivity by reduction of GFAP, NG2, and CHL1 expression. Our current study supports the role of cell cycle activation in the pathophysiology of SCI and by using a clinically relevant treatment model, provides further support for the therapeutic potential of cell cycle inhibitors in the treatment of human SCI.