Effect of chronic adrenalectomy on neuron loss and distribution of sulfated glycoprotein-2 in the dentate gyrus of prepubertal rats

This study extends the unexpected finding of Sloviter et al. (Science, 1989, 243: 535-538) that adrenalectomy (ADX) of young rats casues a loss of granule neurons in the dentate gyrus. In particular, we determined how the vulnerability of dentate granule neurons to the cytocidal effect of ADX is related to the completeness of the ADX and whether sulfated glycoprotein-2, a putative component of programmed cell death, is associated with the death of granule neurons after ADX. We report that 4 months after bilateral ADX of young (150-175 g) rats only ADX rats that had attenuated weight gain and less than 2 ng/ml of serum corticosterone lost granule neurons; whereas as little as 15 ng/ml of serum corticosterone was sufficient to protect granule neurons from cell death. In addition, by immunocytochemistry, SGP-2 was distributed as punctate deposits throughout the molecular layer of the dentate gyrus and in glial cells juxtaposed to surviving neurons in the dentate of ADX rats with a granule cell loss. However, immunoreactivity for SGP-2 was not found in granule neurons that exhibited morphological signs of cellular generation after ADX.     

Sulfated glycoprotein 2: new relationships of this multifunctional protein to neurodegeneration.

Sulfated glycoprotein 2 (SGP-2) from rat, and similar molecules from cow, dog, human, pig, ram and quail are known by 11 or more acronyms. SGP-2 is associated with the responses of brain and other tissues to injury; it and related molecules are also normally secreted by the adrenal gland, the liver and the testes. The mRNA of this protein is found in increased levels in Alzheimer's disease. In rats, after perforant path or excitotoxin lesions, levels of the protein or mRNA are elevated in astrocytes, and also in neurons. In rats, brain SGP-2 is regulated by gonadal and adrenal steroids. However, these increases after brain lesions may relate to a function that is associated with the human protein, namely that of inhibiting complement-mediated cell lysis. Other activities suggested for SGP-2 are lipid transport and cell-cell interactions, which are consistent with sequence data that predict binding of dinucleotides, heparin and lipids. The emerging neurobiology of SGP-2 encompasses the subjects of cell death, synaptic remodelling, neuroendocrinology and neurodegenerative diseases.     

Sulfated glycoprotein-2 is increased in rat hippocampus following entorhinal cortex lesioning.

Thios study showed responses of sulfated glycoprotein-2 (SGP-2) in the rat hippocampus after deafferenting lesion. SGP-2 is a plasma protein that also occurs in many peripheral tissues. In some circumstances, elevations of SGP-2 mRNA are associated with cell degeneration and responses to injury. This study used entorhinal cortex lesions (ECL) to partially deafferent the hippocampus by damaging the perforant path and to induce synaptic remodeling. SGP-2 mRNA is increased in hippocampal astrocytes after ECL. Western blot analysis of soluble hippocampal proteins identified 3 major forms of rat SGP-2 protein: a precursor (61 kDa) and 2 reduced subunits at 39.5 and 35 kDa. These forms increased at 4 days post ECL ipsilaterally to the lesion. By immunocytochemistry (ICC), SGP-2 showed an increased immunoreactivity on the lesioned side by 2 days post ECL that continued through 14 days post ECL. Besides immunopositive astrocytes, punctate immunochemical reaction products occurred among the degenerating fibers of the perforant path. We conclude that changes of SGP-2 protein in the hippocampus after ECL occur roughly in parallel with increases of SGP-2 mRNA. The punctate immuno-deposits could represent secreted SGP-2 and may be useful as a marker for degenerating pathways.     

Sulfated glycoprotein-2 expression increases in rodent brain after transient global ischemia.

Sulfated glycoprotein-2 (SGP-2) is emerging as a prominent marker of neurodegeneration in mammalian brain. Regulation of brain SGP-2 was studied in adult male Wistar rats subjected to 30 min of forebrain ischemia by four vessel occlusion. By 3 days after the ischemic insult, SGP-2 RNA levels were increased two fold in caudate nucleus and hippocampus. SGP-2 protein levels assessed by immunoblots were markedly increased in both brain regions following ischemia. GFAP RNA levels also increased over 5 fold in caudate nucleus and hippocampus following the ischemic insult. Despite significant elevations in GFAP RNA, protein levels of GFAP assessed by immunoblot were only marginally affected. The elevated expression of SGP-2 in rodent brain following this and other experimental lesion paradigms (e.g., excitotoxic lesions, deafferentation) suggest some general involvement of SGP-2 in neurodegeneration and remodelling following neuronal injury.     

Evidence for activation of the terminal pathway of complement and upregulation of sulfated glycoprotein (SGP)-2 in the hypoglossal nucleus following peripheral nerve injury

In a previous study, we found immunoreactivity for complement factors C3, C3d, and C4d, as well as endogenous IgG in the hypoglossal nucleus following hypoglossal nerve transection, suggesting that activation of the complement cascade had taken place in the vicinity of the axotomized motorneurons. In the present study, we found increased immunoreactivity for complement factor C1 and C1q in reactive microglia, indicating an increased potential for initiation of the classical pathway by binding of IgG to C1q. Furthermore, we found immunoreactivity for C9, which contributes to the formation of C5b-9, the final lytic product of the complement cascade close to the axotomized neurons and perineuronal glia. In addition, immunoreactivity and mRNA labeling of sulfated glycoprotein (SGP-2), putative complement inhibitor, was increased in a subpopulation of the axotomized motorneurons. SGP-2 immunoreactivity was also increased in astroglial cells ipsilateral to the nerve injury. The results lend further support to the hypothesis that the complement cascade is activated in the vicinity of axotomized neurons, which in turn may be protected by complement inhibitors. The balance between activation of complement and complement inhibitors might have an impact on the degenerative components of the axon reaction and, in particular, the events leading to nerve cell death.     

Increased expression of caspase 2 in experimental and human temporal lobe epilepsy

Temporal lobe epilepsy (TLE) is often caused by a neurodegenerative brain insult that triggers epileptogenesis, and eventually results in spontaneous seizures, i.e., epilepsy. Understanding the mechanisms of cell death is a key for designing new drug therapies for preventing the neurodegeneration associated with TLE. Here, we investigated the expression of caspase 2, a protein involved in programmed cell death, during the course of epilepsy. We investigated caspase 2 expression in hippocampal samples derived from patients operated on for drug refractory TLE. To understand the evolution of altered-caspase 2 expression during the epileptic process, we also examined caspase 2 expression and activity in the rat hippocampus after status epilepticus-induced acute damage, during epileptogenesis, and after the onset of epilepsy. Caspase 2 expression was enhanced in the hippocampal neurons in chronic TLE patients. In rats, status epilepticus-induced caspase 2 labeling paralleled the progression of neurodegeneration. Proteolytic activation and cleavage of caspase 2 was also detected in the rat brain undergoing epileptogenesis. Our data suggest that caspase 2-mediated programmed cell death participates in the seizure-induced degenerative process in experimental and human TLE. The work was supported by The Academy of Finland, The Kuopio University Foundation, The Neurology Foundation, The North-Savo Regional Fund of the Finnish Cultural Foundation, Sigrid Juselius Foundation, and The Vaajasalo Foundation.     

Apoptosis detection in brain using low-magnification dark-field microscopy.

Apoptosis or programmed cell death is a feature of normal brain development and a response to brain injury. Cells undergoing apoptosis have a characteristic morphology that normally can only be appreciated at high magnification. Using dark-field transmitted light microscopy to examine Nissl-stained material, we detected groups of apoptotic cells at much lower magnifications than often were required in the two injury models we tested. This method was useful for screening entire brain sections to assess regional and global patterns of injury. We predict that this technique in which we detect the clumped chromatin associated with apoptosis can be applied to other types of tissue.     

In situ immunodetection of neuronal caspase-3 activation in Alzheimer disease.

The mechanism by which cells die in Alzheimer disease (AD) is unknown. Several investigators speculate that much of the cell loss may be due to apoptosis, a highly regulated form of programmed cell death. Caspase-3 is a critical effector of neuronal apoptosis and may be inappropriately activated in AD. To address this possibility, we examined cortical and hippocampal brain sections from AD patients, as well as 2 animal models of AD, for in situ evidence of caspase-3 activation. We report here that senile plaques and neurofibrillary tangles in the AD brain are not associated with caspase-3 activation. Furthermore, amyloid beta (A beta) deposition in the APPsw transgenic mouse model of AD does not result in caspase-3 activation despite the ability of A beta to induce caspase-3 activation and neuronal apoptosis in vitro. AD brain sections do, however, exhibit caspase-3 activation in hippocampal neurons undergoing granulovacuolar degeneration. Our data suggests that caspase-3 does not have a significant role in the widespread neuronal cell death that occurs in AD, but may contribute to the specific loss of hippocampal neurons involved in learning and memory.     

Triggers of apoptosis in the immature brain

Apoptosis occurs physiologically in the mammalian brain during the period of the growth spurt, which in human starts in the 3rd trimester of gestation and ends by the third year of life. Environmental factors can interact with programmed cell death mechanisms to increase the number of neurons undergoing apoptosis and thus produce neuropathological sequelae in the brain. In a series of studies it could be shown that classes of drugs which block N-methyl-d-aspartate (NMDA) glutamate receptors, promote γ-aminobutyric-acid (GABA_A) receptor activation or block voltage gated sodium channels, when administered to immature rodents during the period of the brain growth spurt, trigger widespread apoptotic neurodegeneration throughout the developing brain. Studies have also shown that short exposures to non-physiologic oxygen levels can trigger apoptotic neurodegeneration in the brains of infant rodents. Pathomechanisms involved in the proapoptotic action of sedative and anticonvulsant drugs and oxygen include decreased expression of neurotrophins, inactivation of survival signaling proteins, activation of inflammatory cytokines as well as oxidative stress. These findings raise concerns pertaining to the treatment of infants and young children with sedative and anticonvulsant drugs and premature infants with oxygen. The experimental findings imply that new approaches should be developed for patients within these vulnerable age groups.     

Increased cerebellar Purkinje cell numbers in mice overexpressing a human Bcl-2 transgene

The Purkinje cell is a primary organizer in the development of the cerebellum. Purkinje cells may provide positional information cues that regulate afferent innervation, and Purkinje cell target size controls the adult number of afferent olivary neurons and granule cells. While Purkinje cells are necessary for the survival of olivary neurons and granule cells during periods of programmed cell death, little is known about the survival requirements of Purkinje cells in vivo. To determine if Purkinje cells are subject to programmed cell death during development we have analyzed Purkinje cell numbers in two lines of transgenic mice that overexpress a human gene for bcl-2 (Hu-bcl-2). Bcl-2 is a protooncogene that inhibits apoptosis in many cell types. Overexpression of bcl-2 in vitro and in vivo rescues neurons from trophic factor deprivation or naturally occurring cell death. In the mice analyzed in this study, transgene expression is driven by the neuron-specific enolase promoter that is first expressed embryonically in most regions of the brain in one line and postnatally in the second line. We have counted Purkinje cells in three adult control mice, five early overexpressing transgenics, and three late expressing transgenics. The number of Purkinje cells in the Hu-bcl-2 transgenic mice is significantly increased above control numbers, with an increase of 43% in the embryonically overexpressing line and an increase of 27% in the postnatally overexpressing line. Because bcl-2 overexpression has been shown to rescue other neurons from programmed cell death, the increase in Purkinje cell numbers in overexpressing bcl-2 transgenics suggests that Purkinje cells undergo a period of cell death during normal development. © 1996 Wiley-Liss, Inc.     

Common patterns of Bcl-2 family gene expression in two traumatic brain injury models

Cell death/survival following traumatic brain injury (TBI) may be a result of alterations in the intracellular ratio of death and survival factors. Bcl-2 family genes mediate both cell survival and the initiation of cell death. Using lysate RNase protection assays, mRNA expression of the anti-cell death genes Bcl-2 and Bcl-xL, and the pro-cell death gene Bax, was evaluated following experimental brain injuries in adult male Sprague-Dawley rats. Both the lateral fluid-percussion (LFP) and the lateral controlled cortical impact (LCI) models of TBI showed similar patterns of gene expression. Anticell death bcl-2 and bcl-xL mRNAs were attenuated early and tended to remain depressed for at least 3 days after injury in the cortex and hippocampus ipsilateral to injury. Pro-cell death bax mRNA was elevated in these areas, usually following the decrease in anti-cell death genes. These common patterns of gene expression suggest an important role for Bcl-2 genes in cell death and survival in the injured brain. Understanding the regulation of these genes may facilitate the development of new therapeutic strategies for a condition that currently has no proven pharmacologic treatments.     

Corticosterone differentially regulates the bilateral response of astrocyte mRNAs in the hippocampus to entorhinal cortex lesions in male rats.

This study examined the effect of adrenalectomy (ADX) and corticosterone (CORT) replacement on the levels of two astrocyte mRNAs during responses to unilateral entorhinal cortex lesions (ECL) to identify molecular mechanisms involved in glucocorticoid modulation of astrocyte activation following deafferentation. Both glial fibrillary acidic protein (GFAP) and sulfated glycoprotein-2 (SGP-2) mRNA were increased in the ipsilateral hippocampus 4 days following unilateral ECL. In unlesioned ADX rats CORT replacement decreased both messages in the hippocampus. CORT replacement suppressed the ECL-induced increase of GFAP mRNA in the contralateral, but not ipsilateral hippocampus of ADX rats. In contrast, CORT decreased SGP-2 mRNA both ipsi- and contralaterally. It is clear that several regulatory mechanisms are responsible for maintaining a physiological balance of astrocyte activity in the adult brain, and that changes in circuit integrity and the endocrine milieu can alter this balance.     

Apoptosis as a general cell death pathway in neurodegenerative diseases

Neurodegenerative processes are generally characterized by the long-lasting course of neuronal death and the selectivity of the neuronal population or brain structure involved in the lesion. Two main common forms of cell death that have been described in neurons as in other vertebrate tissues i.e., necrosis and apoptosis. Necrosis is the result of cellular "accidents", such as those occurring in tissues subjected to chemical trauma. The necrotizing cells swell, rupture and provoke an inflammatory response. Apoptosis, on the other hand, is dependent on the cell's "decision" to commit suicide and die, and therefore is referred to as "programmed cell death" (PCD). The course of apoptotic death is characterized by a massive morphological change, including cell shrinkage, nuclear (chromosome) condensation and DNA degradation. Activation of PCD in an individual cell is based on its own internal metabolism, environment, developmental background and its genetic information. Such a situation occurs in most of the neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases and amyotrophic lateral sclerosis (ALS). In these pathological situations, specific neurons undergo apoptotic cell death characterized by DNA fragmentation, increased levels of pro-apoptotic genes and "apoptotic proteins" both, in human brain and in experimental models. It is of utmost importance to conclusively determine the mode of cell death in neurodegenerative diseases, because new "anti-apoptotic" compounds may offer a means of protecting neurons from cell death and of slowing the rate of cell degeneration and illness progression.     

原癌基因bcl—2与神经组织程序化细胞死亡

生理或病理条件下发生的细胞程序化死亡是一个级联式基因表达的结果，而比bc1－2原癌基因在程序化细胞死亡中的作用是目前研究的热点。现就近年来bcl－2基因在神经细胞的程序化细胞死亡中的作用、特点及意义等方面作一综述。     

Abnormal Ca(2+) regulation in oligodendrocytes from the dysmyelinating jimpy mouse

Jimpy (jp) is a point mutation in the gene on the X chromosome which codes for the major myelin proteolipid protein. Most oligodendrocytes (OLs) in the jp mouse undergo cell death at the time when they should be actively myelinating. Loss of mature OLs results in severe CNS dysmyelination. Dying jp OLs have the morphology of apoptotic cells but it is not clear how the mutation activates biochemical pathways which lead to programmed death of OLs in jp CNS. There is compelling evidence from a number of systems that high levels of intracellular Ca(2+) ([Ca2+]i) can activate downstream processes which result in both apoptotic and necrotic cell death. To determine whether [Ca2+](i) dysregulation might be involved in the death of jp OLs, we used ratiometric imaging to determine levels of [Ca2+](i) in OLs cultured from jp and normal CNS and in immortalized cell lines derived from jp and normal OLs. Immortalized jp OLs and OLs isolated directly from jp brain both showed a similar elevation in [Ca2+](i) ranging from 60% to 150% over control values. A higher baseline [Ca2+](i) in jp OLs might increase their vulnerability to other insults due to abnormal protein processing or changes in signaling pathways which act as a final trigger for cell death.     

To die or not to die, does it change the function? Behavior of transgenic mice reveals a role for developmental cell death

In humans, perturbations in the developmental neuronal death leading to an excess of neurons could be associated with developmental neuropsychiatric disorders. Hu-bcl-2 transgenic mice appear to be a valuable tool to study the functional role of developmental programmed cell death. Indeed, the over-expression of the anti-apoptotic gene bcl-2 decreases developmental neuronal death and Hu-bcl-2 mice present supernumerary neurons in several brain regions. A detailed behavioral analysis of these mice revealed selective deficits. Hu-bcl-2 mice have normal vision, general activity and motor skills. Only the most complex behavior like anxiety and learning abilities are impaired in these mice.     

Genetic Manipulation of Cell Death and Neuroplasticity Pathways in Traumatic Brain Injury

Traumatic brain injury (TBI) initiates a complex cascade of secondary neurodegenerative mechanisms contributing to cell dysfunction and necrotic and apoptotic cell death. The injured brain responds by activating endogenous reparative processes to counter the neurodegeneration or remodel the brain to enhance functional recovery. A vast array of genetically altered mice provide a unique opportunity to target single genes or proteins to better understand their role in cell death and endogenous repair after TBI. Among the earliest targets for transgenic and knockout studies in TBI have been programmed cell death mediators, such as the Bcl-2 family of proteins, caspases, and caspase-independent pathways. In addition, the role of cell cycle regulatory elements in the posttraumatic cell death pathway has been explored in mouse models. As interest grows in neuroplasticity in TBI, the use of transgenic and knockout mice in studies focused on gliogenesis, neurogenesis, and the balance of growth-promoting and growth-inhibiting molecules has increased in recent years. With proper consideration of potential effects of constitutive gene alteration, traditional transgenic and knockout models can provide valuable insights into TBI pathobiology. Through increasing sophistication of conditional and cell-type specific genetic manipulations, TBI studies in genetically altered mice will be increasingly useful for identification and validation of novel therapeutic targets.     

Apoptosis and schizophrenia: is the tumour suppressor gene, p53, a candidate susceptibility gene?

This paper reviews the six published incidence studies of the relative risk of cancer in patients with schizophrenia compared with the general population. These studies used: incidence data, register case ascertainment, and controlled for age and sex. It is concluded that schizophrenia is associated with a lower risk of developing cancer. The role of apoptosis (programmed cell death) in cancer and brain development is briefly described. The possibility is explored that increased apoptosis may account for neurodevelopmental abnormalities as well as tumour resistance associated with schizophrenia. The authors propose that p53, a tumour suppressor gene central to regulation of apoptosis, should be investigated as a candidate susceptibility gene in schizophrenia.