Background Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND), including memory dysfunction, continue to be a major clinical manifestation of HIV type-1 (HIV-1) infection. Viral proteins released by infected glia are thought to be the principal triggers of inflammation and bystander neuronal injury and death, thereby driving key symptomatology of HAND. Methods We used a GFAP-driven, doxycycline (DOX)-inducible HIV-1 Tat (transactivator of transcription) transgenic mouse model and examined structure-function relationships in hippocampal pyramidal CA1 neurons using morphologic (Golgi-silver impregnations, immunohistochemistry, TUNEL detection, synaptic protein markers, electron microscopy), electrophysiological (long-term potentiation (LTP)), and behavioral (Morris water maze, fear-conditioning) approaches. Results Tat induction caused a variety of different inclusions in astrocytes characteristic of lysosomes, autophagic vacuoles, and lamellar bodies, which were typically present within distal cytoplasmic processes. In pyramidal CA1 neurons, Tat induction reduced the number of apical dendritic spines, while disrupting the distribution of synaptic proteins (synaptotagmin 2 and gephyrin) associated with inhibitory transmission, but with minimal dendritic pathology and no evidence of pyramidal neuron death. Electrophysiological assessment of excitatory postsynaptic field potential (fEPSP) at Schaffer collateral/commissural fiber-CA1 synapses showed near total suppression of LTP in mice expressing Tat. The loss in LTP coincided with disruptions in learning and memory. Conclusion Tat expression in the brain results in profound functional changes in synaptic physiology and in behavior that are accompanied by only modest structural changes and minimal pathology. Tat likely contributes to HAND by causing molecular changes that disrupt synaptic organization, with inhibitory presynaptic terminals containing synaptotagmin 2 appearing especially vulnerable.

Sexual dimorphism of neurons and astrocytes has been demonstrated in different centers of the brain, but sexual dimorphism of oligodendrocytes and myelin has not been examined. We show, using immunocytochemistry and in situ hybridization, that the density of oligodendrocytes in corpus callosum, fornix, and spinal cord is 20 -40% greater in males compared with females. These differences are present in young and aged rodents and are independent of strain and species. Proteolipid protein and carbonic anhydrase-II transcripts, measured by real-time PCR, are approximately two to three times greater in males. Myelin basic protein and 2Ј,3Ј-cyclic nucleotide 3Ј-phosphodiesterase, measured by Western blots, are 20 -160% greater in males compared with females. Surprisingly, both generation of new glia and apoptosis of glia, including oligodendrocytes, are approximately two times greater in female corpus callosum. These results indicate that the lifespan of oligodendrocytes is shorter in females than in males. Castration of males produces a female phenotype characterized by fewer oligodendrocytes and increased generation of new glia. These findings indicate that exogenous androgens differentially affect the lifespan of male and female oligodendrocytes, and they can override the endogenous production of neurosteroids. The data imply that turnover of myelin is greater in females than in males. -Calpain, a protease upregulated in degeneration of myelin, is dramatically increased at both transcriptional and translational levels in females compared with males. These morphological, molecular, and biochemical data show surprisingly large differences in turnover of oligodendrocytes and myelin between sexes. We discuss the potential significance of these differences to multiple sclerosis, a sexually dimorphic disease, whose progression is altered by exogenous hormones.

Peroxisome proliferator–activated receptors (PPARs) are ligand‐activated transcription factors of the nuclear hormone receptor superfamily that have been described as master genes that switch cells from an undifferentiated phenotype to a differentiated phenotype. In the present investigation, we examined the possibility that ligands for PPARs are potent activators of oligodendrocyte (OL) differentiation and/or proliferation. Primary glial cultures and enriched OL cultures of neonatal mouse cerebra were treated with three different PPAR agonists: a PPAR γ–selective agonist, a PPAR δ–selective agonist, and a pan agonist selective for both PPAR γ and δ. Treatment with PPAR γ agonist does not have an effect on the differentiation of OLs; however, PPAR δ agonist and the pan agonist treatment accelerates the differentiation of OLs within 24 h of application in mixed glial cultures. The number of OLs with processes and huge membrane sheets increases two‐ to threefold in both groups. The increase in the size of the sheets is also mirrored by changes in the intensity and distribution of myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs. As compared to controls, the PPAR δ agonist–treated groups contain more OLs that have MBP and PLP mRNA extending into distal processes. These results indicate that PPAR δ plays a significant role in the maturation of OLs and regulates the size of OL sheets. BrdU immunostaining reveals that these agonists do not significantly stimulate proliferation of OLs expressing glycolipids. The studies in enriched OL cultures reproduce the effects of the PPAR agonists seen in the mixed glial cultures, indicating that the effect of the PPAR agonists is directly on the OLs and not via astrocytes. In the enriched cultures, the total number of OLs increases significantly in the PPAR δ agonist–treated groups, but BrdU immunostaining does not show an increased proliferation of cells. These findings suggest that PPAR δ increases the survival of cells and/or prevents cell death in enriched cultures. Although PPAR δ is expressed in various cell types, its role as a factor in the transcriptional regulation of OL differentiation has not been explored. We show for the first time that a ligand that serves as an agonist for PPAR δ activates the program of OL differentiation in primary and enriched OL cultures. GLIA 33:191–204, 2001. © 2001 Wiley‐Liss, Inc.

Electron microscopy and 3H-thymidine autoradiographic techniques were used to study the fine structure of proliferating cells in developing rat optic nerve. Before the closure of the optic canal almost all of the cells incorporating radioactive thymidine are ventricular cells, but after closure (16 days of gestation) the vast majority are differentiating astroblasts or oligodendroblasts. Labeled astroblasts show a range in their degree of differentiation; some cells lack 90 A cytoplasmic filaments while others have glial filaments and abundant cytoplasmic organelles. In contrast to astroblasts, all of the labeled oligodendroblasts are in the early stages of differentiation. The proliferation of oligodendroblasts starts at five days postnatal, approximately a day or two before the onset of myelination.During myelinogenesis a few of the labeled oligodendroblasts show presumptive connections to myelin sheaths. Microglial cells do not appear to play a major role in gliogenesis since they form less than 2% of all the labeled cells. The results of this study indicate that astroblasts and oligodendroblasts, rather than undifferentiated glioblasts, are the major source of macroglia. The finding that proliferating glia are in the processof differentiation agrees with recent studies which show that differentiated cells can divide.

Using primary cultures of oligodendrocyte progenitors isolated from male and female neonatal rodent brains, we observed more oligodendrocytes in female-derived compared to male-derived cultures. To determine whether the observed differences were due to a differential effect of sex hormones on proliferation, we treated cultures with increasing doses of 17β-estradiol, testosterone or progesterone and labeled cells with bromodeoxyuridine to identify cells in S phase. Treatment with 17β-estradiol, but not progesterone or testosterone, delayed the exit of oligodendrocyte progenitor cells from the cell cycle. In addition, 17β-estradiol treatment enhanced membrane sheet formation, while progesterone increased cellular branching. Interestingly, the estrogen modulator tamoxifen mimicked the effect of 17β-estradiol on cell cycle exit, but not on membrane formation. Immunocytochemical localization of estrogen receptors (ERs) showed ERβ mainly localized to the cytoplasm of oligodendrocytes, suggesting that the effect of 17β-estradiol on membrane formation could be mediated by interaction with this receptor. We conclude that sex steroids differentially regulate oligodendrocyte progenitor number and myelin formation, possibly contributing to gender-specific differences in repair.

Neonatal periventricular white matter injury is a major contributor to chronic neurologic dysfunction. In a neonatal rat stroke model, myelin basic protein (MBP) immunostaining reveals acute periventricular white matter injury. Yet, the extent to which myelin proteins can recover after neonatal hypoxicischemic injury is unknown. We developed a quantitative method to correlate the severity of the hypoxic-ischemic insult with the magnitude of loss of MBP immunostaining. Seven-day-old (P7) rats underwent right carotid ligation, followed by exposure to 8% oxygen for 1, 1.5, 2, or 2.5 h. On both P12 and P21, white matter integrity was evaluated by densitometric analysis of MBP immunostaining, and the amount of tissue injury was evaluated by morphometric measurements of cerebral hemisphere areas. The most severe hypoxic-ischemic insults (2.5 h) elicited marked reductions in MBP immunostaining ipsilaterally on both P12 and P21. In contrast, in mildly lesioned animals (1.5 h), MBP immunostaining was reduced ipsilaterally on P12, but 2 wk after lesioning, on P21, there was a substantial restoration of MBP immunostaining. The restoration in MBP immunostaining could reflect either functional recovery of injured oligodendroglia or proliferation and maturation of oligodendroglial precursors. Our data demonstrate that quantitative measurement of MBP immunostaining provides a sensitive indicator of acute oligodendroglial injury. Most importantly, we show that in this neonatal rodent stroke model, restoration of myelin proteins occurs after moderate, but not after more severe, cerebral hypoxia-ischemia. Considerable clinical and experimental data demonstrate that immature oligodendroglia are highly susceptible to HI injury (1-5). There is also growing recognition that oligodendroglial injury in the immature nervous system may have profound adverse effects on neuronal development (6). Among the mechanisms implicated as contributing to the vulnerability of immature oligodendroglia to HI are increased susceptibility to excitotoxicity (1, 7), oxidative stress (2), and inflammation (8 -9), and propensity for induction of apoptosis (10).In a widely used neonatal rat stroke model, elicited by unilateral carotid artery ligation and timed exposure to moderate hypoxia (8% oxygen) in 7-d-old (P7) rats, ipsilateral white matter injury occurs, together with more widespread tissue injury (11)(12)(13)(14)(15). In this model, lengthening the duration of hypoxic exposure results in a progressive increase in the severity of brain damage (12); the temporal threshold to elicit tissue injury is at approximately 1.5 h, and more prolonged HI (2-2.5 h) commonly elicits extensive neuronal loss in the ipsilateral cortex, hippocampus, striatum, and thalamus (13). Additional neuropathologic features of the HI lesion include an acute and sustained microglial and monocyte infiltrate (13, 14), reactive gliosis (13, 15), and the evolution of cortical cavitary infarcts (13).Although the original description of histopathology in this model documented the vulner...

Neonatal hypoxic-ischemic (HI) white matter injury is a major contributor to chronic neurological dysfunction. Immature oligodendrocytes (OLGs) are highly vulnerable to HI injury. As little is known about in vivo OLG repair mechanisms in neonates, we studied whether new OLGs are generated after HI injury in P7 rats. Rats received daily BrdU injections at P12-14 or P21-22 and sacrificed at P14 to study the level of cell proliferation or at P35 to permit dividing OLG precursors to differentiate. In P14 HI-injured animals, the number of BrdU+ cells in the injured hemisphere is consistently greater than controls. At P35, sections were double-labeled for BrdU and markers for OLGs, astrocytes, and microglia. Double-labeled BrdU+/myelin basic protein+ and BrdU+/carbonic anhydrase+ OLGs are abundant in the injured striatum, corpus callosum, and the infarct core. Quantitative studies show four times as many OLGs are generated from P21-35 in HI corpora callosa than controls. Surprisingly, the infarct core contains many newly generated OLGs in addition to hypertrophied astrocytes and activated microglia. These glia and non-CNS cells may stimulate OLG progenitor proliferation or induce their migration. At P35, astrogliosis and microgliosis are dramatic ipsilaterally but only a few microglia and some astrocytes are BrdU+. This finding indicates microglial and astrocytic hyperplasia occurs shortly after HI but before the P21 BrdU injections. Although the neonatal brain undergoes massive cell death and atrophy the first week after injury, it retains the potential to generate new OLGs up to 4 weeks after injury within and surrounding the infarct.

No abstract