Gye Sun Jeon

Cerebellar alterations induced by chronic hypoxia: an immunohistochemical study using a chick embryonic model

A model of fetal aerogenic hypoxia was developed in which fertilized chicken eggs were half-painted with melted wax and incubated under normal conditions. The cerebellum of the hypoxic chick embryos at a later stage of development (E18-20) was analyzed immunochemically. Hypoxic insult resulted in considerable neurocytological deficits of the Purkinje cells and altered glial fibrillary acid protein (GFAP) immunoreactivity in the fetal cerebellum. Purkinje cells in the hypoxic embryos were marked by small cell size, poorly developed dendrites, low cell density, deletion and ectopia. On the other hand, enhanced GFAP immunoreactivity was found in astrocytes and Bergmann glia of the hypoxic embryos. Our results indicate that chronic hypoxia in the chick fetus can cause severe disorders of neuronal development as well as glial activation. We suggest that our hypoxic model of chick embryos could be an accessible animal model for further elucidating fetal hypoxia.

Glial expression of the 90-kDa heat shock protein (HSP90) and the 94-kDa glucose-regulated protein (GRP94) following an excitotoxic lesion in the mouse hippocampus

Heat shock proteins (HSPs) are immediately expressed in neuronal and glial cells under various stressful conditions and play a protective role through molecular chaperones. Although several studies have been focused on the expression of HSPs, little is known about HSP90s expression in glial cells under neuropathological conditions. In this study, we evaluated the expression pattern of the glial cell‐related HSP90 and GRP94 proteins, following the induction of an excitotoxic lesion in the mouse brain. Adult mice received an intracerebroventricular injection of kainic acid; the brain tissue was then analyzed immunohistochemically for HSPs and double labeling using glial markers. HSPs expression was quantified by Western blot analysis. Excitotoxic damage was found to cause pyramidal cell degeneration in the CA3 region of the hippocampus. In the injured hippocampus, reactive microglia/macrophages expressed HSP90 from 12 h until 7 days postlesion (PL), showing maximal levels at day 1. In parallel, hippocampal reactive astrocytes showed the expression of GRP94 from 12 h until 7 days PL. In general, HSPs expression was transient, peaked at 1–3 days PL and reached basal levels by day 7. For the first time, our data demonstrate the injury‐induced expression of HSP90 and GRP94 in glial cells, which may contribute to the mechanism of glial cell protection and adaptation in response to damage, thereby playing an important role in the evolution of the glial response and the excitotoxic lesion outcome. HSP90 may provide antioxidant protective mechanisms against microglia/macrophages, whereas GRP94 may stabilize the astroglial cytoskeleton and participate in astroglial antioxidant mechanisms.

Relative sparing of calretinin containing neurons in the substantia nigra of 6-OHDA treated rat Parkinsonian model

A certain calcium binding protein (CaBP) has been known to exert a neuroprotective effect in various neurodegenerative diseases. Using the 6-OHDA induced rat Parkinsonian model, we examined if calretinin (CR), one of CaBP family, could play the similar role in the Parkinsons disease because CR is profusely localized in dopaminergic neurons of the substantia nigra pars compacta (SNPC) of the rat. Employing immunohistochemical analyses, we found that the survival rate of CR neurons was significantly higher than that of tyrosine hydroxylase (TH) neurons in the SNPC of the Parkinsonian rat. Furthermore double-labeled fluorescent microscopy revealed that almost all surviving TH neurons were also positive to CR. Our data suggest that CR-positive neurons are less vulnerable to 6-OHDA and CR in the dopaminergic neurons may have a protective function for survival of these neurons in the experimentally induced Parkinsonian rat.

Potential Effect of S-Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

Aggregation of misfolded protein and resultant intracellular inclusion body formation are common hallmarks of mutant superoxide dismutase (mSOD1)-linked familial amyotrophic lateral sclerosis (FALS) and have been associated with the selective neuronal death. Protein disulfide isomerase (PDI) represents a family of enzymatic chaperones that can fold nascent and aberrant proteins in the endoplasmic reticulum (ER) lumen. Recently, our group found that S-nitrosylated PDI could contribute to protein misfolding and subsequent neuronal cell death. However, the exact role of PDI in the pathogenesis of ALS remains unclear. In this study, we propose that PDI attenuates aggregation of mutant/misfolded SOD1 and resultant neurotoxicity associated with ER stress. ER stress resulting in PDI dysfunction therefore provides a mechanistic link between deficits in molecular chaperones, accumulation of misfolded proteins, and neuronal death in neurodegenerative diseases. In contrast, S-nitrosylation of PDI inhibits its activity, increases mSOD1 aggregation, and increases neuronal cell death. Specifically, our data show that S-nitrosylation abrogates PDI-mediated attenuation of neuronal cell death triggered by thapsigargin. Biotin switch assays demonstrate S-nitrosylated PDI both in the spinal cords of SOD1 (G93A) mice and human patients with sporadic ALS. Therefore, denitrosylation of PDI may have therapeutic implications. Taken together, our results suggest a novel strategy involving PDI as a therapy to prevent mSOD1 aggregation and neuronal degeneration. Moreover, the data demonstrate that inactivation of PDI by S-nitrosylation occurs in both mSOD1-linked and sporadic forms of ALS in humans as well as mice.

Constitutive expression of c-myb mRNA in the adult rat brain

In this study, we demonstrated the c-myb mRNA expression in the adult rat brain using an in situ hybridization technique. We found c-myb mRNA signals in the various regions of the forebrain and midbrain including the cerebral cortex, thalamus, hippocampus, hypothalamus, superior and inferior colliculi and central gray. In the cerebellum, a diffuse signal was found in the granular layer while some positive cells were detected in the molecular layer as well. In addition, a number of cells showed intense signals in many nuclei of the medulla oblongata. The constitutive expression of c-myb mRNA in the different kinds of neural cells suggests that this gene might be involved in the normal function of these neurons.

ATRX induction by mutant huntingtin via Cdx2 modulates heterochromatin condensation and pathology in Huntington's disease

Aberrant chromatin remodeling is involved in the pathogenesis of Huntingtons disease (HD) but the mechanism is not known. Herein, we report that mutant huntingtin (mtHtt) induces the transcription of alpha thalassemia/mental retardation X linked (ATRX), an ATPase/helicase and SWI/SNF-like chromatin remodeling protein via Cdx-2 activation. ATRX expression was elevated in both a cell line model and transgenic model of HD, and Cdx-2 occupancy of the ATRX promoter was increased in HD. Induction of ATRX expanded the size of promyelocytic leukemia nuclear body (PML-NB) and increased trimethylation of H3K9 (H3K9me3) and condensation of pericentromeric heterochromatin, while knockdown of ATRX decreased PML-NB and H3K9me3 levels. Knockdown of ATRX/dXNP improved the hatch rate of fly embryos expressing mtHtt (Q127). ATRX/dXNP overexpression exacerbated eye degeneration of eye-specific mtHtt (Q127) expressing flies. Our findings suggest that transcriptional alteration of ATRX by mtHtt is involved in pericentromeric heterochromatin condensation and contributes to the pathogenesis of HD.

Differential expression of c-Ret in motor neurons versus non-neuronal cells is linked to the pathogenesis of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by selective degeneration of motor neurons throughout the central nervous systems. Non-cell autonomous damage induced by glial cells is linked to the selective susceptibility of motor neurons in ALS, but the mechanisms underlying this phenomenon are not known. We found that the expression of non-phosphorylated and phosphorylated forms (tyrosine (Tyr) residue 905, 1016, and 1062) of c-Ret, a member of the glial cell line-derived neurotrophic factor (GDNF) receptor, are altered in motor neurons of the lumbar spinal cord in ALS transgenic (G93A) mice and ALS (G93A) cell line models. Phosphorylated forms of c-Ret were colocalized with neurofilament aggregates in motor neurons of ALS mice. Consistent with the in vivo data, levels of non-phosphorylated and phosphorylated c-Ret (Tyr 905, 1016, and 1062) were decreased by oxidative stress in motor neuronal cells (NSC-34). Non-phosphorylated and phosphorylated forms of c-Ret immunoreactivity were markedly elevated in active microglia of ALS mice. Our findings suggest that constitutive oxidative stress modulates c-Ret function, thereby reducing GDNF signaling in motor neurons. Furthermore, the induction of c-Ret expression in microglia may contribute to non-cell autonomous cell death of motor neurons by available GDNF in ALS.

Age-related changes in glycogen synthase kinase 3β (GSK3β) immunoreactivity in the central nervous system of rats

Although glycogen synthase kinase 3beta (GSK3beta) is emerging as a prominent drug target in the treatment of neurodegenerative diseases such as Alzheimers disease (AD) and stroke, very little is known about age-related changes in GSK3beta expression and GSK3beta phosphorylation. Therefore, we examined age-related changes in immunoreactivities for GSK3beta and phosphorylated GSK3beta (pGSK3beta) in the central nervous system. In aged rats, there were significant increases in GSK3beta immunoreactivity in the cell bodies and processes of pyramidal cells in most cortical regions. GSK3beta immunoreactivity was also significantly increased in the pyramidal layer of CA1-3 regions, and the granule cell layer of dentate gyrus. Age-related increases were prominent in lateral septal nuclei, compared to the medial septal nuclei. Interestingly, both GSK3beta and pGSK3beta was increased in the prefrontal cortex, while GSK3beta and pGSK3beta was differentially localized in the cerebellar cortex. The first demonstration of age-related alterations in immunoreactivities for GSK3beta and pGSK3beta in the basal forebrain area and cholinergic projection targets may provide useful data for investigating the pathogenesis of age-related neurodegenerative diseases including AD.

Deregulation of BRCA1 Leads to Impaired Spatiotemporal Dynamics of γ-H2AX and DNA Damage Responses in Huntington’s Disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder of mid-life onset characterized by involuntary movements and progressive cognitive decline caused by a CAG repeat expansion in exon 1 of the Huntingtin (Htt) gene. Neuronal DNA damage is one of the major features of neurodegeneration in HD, but it is not known how it arises or relates to the triplet repeat expansion mutation in the Htt gene. Herein, we found that imbalanced levels of non-phosphorylated and phosphorylated BRCA1 contribute to the DNA damage response in HD. Notably, nuclear foci of γ-H2AX, the molecular component that recruits various DNA damage repair factors to damage sites including BRCA1, were deregulated when DNA was damaged in HD cell lines. BRCA1 specifically interacted with γ-H2AX via the BRCT domain, and this association was reduced in HD. BRCA1 overexpression restored γ-H2AX level in the nucleus of HD cells, while BRCA1 knockdown reduced the spatiotemporal propagation of γ-H2AX foci to the nucleoplasm. The deregulation of BRCA1 correlated with an abnormal nuclear distribution of γ-H2AX in striatal neurons of HD transgenic (R6/2) mice and BRCA1+/− mice. Our data indicate that BRCA1 is required for the efficient focal recruitment of γ-H2AX to the sites of neuronal DNA damage. Taken together, our results show that BRCA1 directly modulates the spatiotemporal dynamics of γ-H2AX upon genotoxic stress and serves as a molecular maker for neuronal DNA damage response in HD.

Oligodendroglia in the avian retina: immunocytochemical demonstration in the adult bird.

Immunohistochemical techniques were used in conjunction with an avian‐specific probe for oligodendrocyte (OLG) marker, the antibody for transferrin binding protein (TfBP), to study the characteristics and distribution of OLGs in the retina of chickens and quails. For comparison, other antibodies such as myelin basic protein, Rip, and those for labeling Müller cells and microglia were used. A large population of OLGs was found to be distributed throughout the retina, with the distinct pattern of a central‐to‐peripheral gradient. It was possible to detect a spectrum of OLG morphology that bore a resemblance to the subtype of the mammalian central nervous system. In addition to these mature OLGs, limited numbers of TfBP‐positive (TfBP+) cells with the morphology of immature OLGs were found in the immediate vicinity of the optic head. The majority of OLGs appeared in the ganglion cell layer throughout the retina, whereas OLGs in the nerve fiber layer were seen mainly in the central zone of the retina, near the optic nerve head. Double‐labeling experiments showed that OLGs were associated with myelin only in the central region, where the majority of retinal OLGs occurred, but not toward the periphery of the retina. The present study is the first comprehensive analysis of the morphological features and spatial distribution of OLGs in the adult avian retina and provides in vivo evidence for the existence of a substantial population of both mature and immature OLGs in the retina of adult birds. The putative functions of TfBP+ OLGs including myelination and the tropic role of the ganglion cells are discussed in conjunction with the physical properties of TfBP and structural characteristics of the avascular retina of birds. J. Neurosci. Res. 65:173–183, 2001.