Susceptibility to cell death induced by mutant SV40 T-antigen correlates with Purkinje neuron functional development

Purkinje cells are uniquely susceptible to a number of physical, chemical, and genetic insults both during development and in the mature state. We have previously shown that when the postmitotic state of murine Purkinje cells is altered by inactivation of the retinoblastoma tumor susceptibility protein (pRb), immature as well as mature Purkinje cells undergo apoptosis. DNA synthesis and neuronal loss are induced in postmitotic Purkinje cells dependent upon the pRb-binding portion of SV40 large T antigen (T-ag). In the present study, Purkinje cell targeting of a mutant T-ag, PVU, which does not bind pRb, reveals disparate cerebellar phenotypes dependent upon temporal differences in transgene expression. Strong embryonic and postnatal transgene expression in three lines alters Purkinje cell development and function during the second postnatal week, causing ataxia without Purkinje cell loss. In contrast, two other transgenic lines reveal that PVU T-ag expression following normal Purkinje cell maturation causes rapid Purkinje cell degeneration. The second and third postnatal weeks of cerebellar development, which include the major period of synaptogenesis, appear to be the defining stage for the two PVU-induced phenotypes. These data indicate that Purkinje cell death susceptibility varies with developmental stage.     

The olivocerebellar projection mediates ibogaine-induced degeneration of Purkinje cells: a model of indirect, trans-synaptic excitotoxicity

Ibogaine, an indole alkaloid that causes hallucinations, tremor, and ataxia, produces cerebellar neurotoxicity in rats, manifested by degeneration of Purkinje cells aligned in narrow parasagittal bands that are coextensive with activated glial cells. Harmaline, a closely related alkaloid that excites inferior olivary neurons, causes the same pattern of Purkinje cell degeneration, providing a clue to the mechanism of toxicity. We have proposed that ibogaine, like harmaline, excites neurons in the inferior olive, leading to sustained release of glutamate at climbing fiber synapses on Purkinje cells. The objective of this study was to test the hypothesis that increased climbing fiber activity induced by ibogaine mediates excitotoxic Purkinje cell degeneration. The inferior olive was pharmacologically ablated in rats by a neurotoxic drug regimen using 3-acetylpyridine, and cerebellar damage attributed to subsequent administration of ibogaine was analyzed using immunocytochemical markers for neurons and glial cells. The results show that ibogaine administered after inferior olive ablation produced little or no Purkinje cell degeneration or glial activation. That a lesion of the inferior olive almost completely prevents the neurotoxicity demonstrates that ibogaine is not directly toxic to Purkinje cells, but that the toxicity is indirect and dependent on integrity of the olivocerebellar projection. We postulate that ibogaine-induced activation of inferior olivary neurons leads to release of glutamate simultaneously at hundreds of climbing fiber terminals distributed widely over the surface of each Purkinje cell. The unique circuitry of the olivocerebellar projection provides this system with maximum synaptic security, a feature that confers on Purkinje cells a high degree of vulnerability to excitotoxic injury.     

An immunohistochemical study of Purkinje cells in a case of hereditary cerebellar cortical atrophy

We report an immunohistochemical study of Purkinje cells located in the molecular layer (ectopic Purkinje cells) and in the Purkinje cell layer (orthotopic Purkinje cells) of a patient who died young and had been diagnosed with hereditary cerebellar cortical atrophy from both clinical and neuropathological findings. All the ectopic and orthotopic Purkinje cells were immunoreactive with an anti-calbindin-D antibody, confirming that these stained cells were Purkinje cells. The perikarya of some ectopic and orthotopic Purkinje cells were stained by an antibody to phosphorylated neurofilament. In contrast, Purkinje cells of three normal controls did not react with this antibody. This finding of an abnormal accumulation of phosphorylated neurofilaments in the perikarya may be due to abnormal phosphorylation. Moreover, the regions around the cell bodies of some ectopic and orthotopic Purkinje cells were intensely immunoreactive with an antibody to synaptophysin, which suggests an abnormal increase in presynaptic terminals. It is suggested that ectopia of Purkinje cells, accumulation of phosphorylated neurofilament in the perikarya and an abnormal increase in presynaptic terminals around the soma of the Purkinje cells may be relevant to the pathophysiology of Purkinje cell degeneration in this case. In addition, the relationship between phosphorylated neurofilament and synaptophysin reactivity is discussed.     

Paraneoplastic cerebellar degeneration--characterization of anti-Yo antibody and underlying cancer

A group of patients with paraneoplastic cerebellar degeneration (PCD) have shown to produce autoantibody to both neurons and tumor cells (anti-Yo antibody). More than 60% of these patients have shown neurological symptoms and anti-Yo antibody production before the underlying cancers were found, which suggests that the test for anti-Yo antibody is important for the early detection and treatment of cancer. Originally, anti-Yo antibody has been characterized as 1) labelling the cytoplasm of cerebellar Purkinje cells immunohistochemically, 2) binding to the 62 and 34kDa bands on immunoblots of Purkinje cell extracts, 3) being present in female patients with gynecological or breast cancers. Recently, the common binding-epitope of anti-Yo antibody has been reported as leucine-zipper protein. In order to detect the anti-Yo antibody precisely, we examined the immunohistochemical and western blot characters of the recombinant leucine-zipper protein-reactive (anti-Yo) antibody. The results were, 1) sera containing leucine-zipper protein-reactive antibody labels both cerebellar Purkinje cells but some sera might contain other antibodies together with anti-Yo that confuse the immunostaining character of anti-Yo antibody, 2) the antibody binds to 58 kDa band and sometimes co-binds to 34kDa on immunoblots of cerebellar tissue extracts. The underlying cancers are mainly adenocarcinoma in the ovary, fallopian tube, uterus, or breast but occasionally large cel lung and bile duct cancers have been found. Interestingly, a male patient had an antibody similar in character to be anti-Yo antibody immunohistochemically and on immunoblots, that did not recognize leucine-zipper protein and the underlying carcinoma was small cell lung cancer. These results suggest that 1) the diagnosis of anti-Yo antibody should be based on the antibody's reactivity with leucine-zipper protein, 2) some sera with the anti-Yo antibody label other tissues besides the Purkinje cell cytoplasm because of the co-existence of other antibodies seen immunohistochemically and on immunoblots, 3) the search for underlying cancers should not be limited to gynecological or breast carcinomas.     

Transplanted neurons alter the course of neurodegenerative disease in Lurcher mutant mice

Embryonic cerebellar, neocortical, and striatal tissues derived from NSE-LacZ transgenic mice were transplanted into the right cerebellar hemisphere of 8- to 10-day-old Lurcher or wild-type mice. Host mice survived for 30-90 days and the transplanted tissue was examined by light microscopy using Nissl staining, X-gal histochemistry, and immunohistochemistry for calcium binding protein and glutamic acid decarboxylase. Transplantation of cerebellar tissue, but not neocortical or striatal progenitors, resulted in robust infiltration of the lurcher mutant host cerebellar cortex by transgenic Purkinje neurons. Deep to the infiltrated molecular layer, the host granular layer was thicker and denser than the mutant granular layer, but transgenic cells did not contribute to the spared granular layer. The host inferior olivary complex consistently exhibited a noticeable bilateral asymmetry in Nissl-stained sections. A quantitative analysis of the olivary complex was performed in 10 90-day-old host mice. The results indicate that the left inferior olivary complex of 90-day-old host mice contained more neurons than the right inferior olive of the host mice and contained more neurons than was observed in 90-day-old Lurcher control mice. Analysis by olivary subdivision indicates that increased neuron numbers were present in all subdivisions of the host left inferior olive. These studies confirm the specific attractive effect of the mutant cerebellar cortex on transplanted Purkinje neuron progenitors and indicate that neural transplants may survive the neurodegenerative period to interact with developing host neural systems. The unilateral rescue of Lurcher inferior olivary neurons in cerebellar transplant hosts indicates that transplanted neurons may interact with diseased host neural circuits to reduce transneuronal degeneration in the course of a neurodegenerative disease.     

Estimating the costs of hip fracture and potential savings

Shaker mutant rats are characterized by the adult-onset degeneration of cerebellar anterior lobe Purkinje cells and temporally correlated development of ataxia and tremor. Normal E-13 Purkinje cells were transplanted into the anterior cerebellum in adult shaker mutant rats to study donor/host interactions in an animal with adult-onset heredodegeneration. Donor Purkinje cells from extraparenchymal transplant sites migrated radially into the host molecular layer and differentiated. Donor Purkinje cell dendrites expanded to fill the host molecular layer, spinous processes were apparent, and axonal projections into the host gray and white matter were observed. Donor Purkinje cells remaining in the extraparenchymal transplant sites differentiated if they were located relatively close to the host cerebellum. Donor Purkinje cells located intraparenchymally in the host white matter or granule cell layer survived, but were stunted in their development. The orthogonal movement of donor Purkinje cells away from transplant sites in the host cerebellum was spatially restricted. The findings from this study indicate that host cerebellar cortex with adult-onset heredodegeneration of Purkinje cells supports the survival and differentiation of transplanted normal embryonic Purkinje cells.     

Regulated ectopic expression of cyclin D1 induces transcriptional activation of the cdk inhibitor p21 gene without altering cell cycle progression

Cyclin D1 plays a key regulatory role during the G1 phase of the cell cycle and its gene is amplified and overexpressed in many cancers. To address the relationship between cyclin D1 and other cell cycle regulatory proteins, we established human glioma and rodent fibroblast cell lines in which cyclin D1 expression could be regulated ectopically with tetracycline. In both of these cell lines, we found that ectopic expression of cyclin D1 in asynchronously growing cells was accompanied by increased levels of the p53 tumor suppressor protein and the cyclin/cdk inhibitor p21. Despite the induction of these cell cycle inhibitory proteins, cyclin D1-associated cdk kinase remained activated and the cells grew essentially like that of the parent cells. Although growth parameters were unchanged in these cells, morphological changes were clearly identifiable and anchorage independent growth was observed in NIH3T3 cells. In a first step toward elaborating the mechanism for cyclin D1-mediated induction of p21 gene expression we show that co-expression of E2F-1 and DP-1 can specifically transactivate the p21 promoter. In support of these findings and a direct effect of E2F on induction of p21 gene expression a putative E2F binding site was identified within the p21 promoter. In summary, our results demonstrate that ectopic expression of cyclin D1 can induce gene expression of the cdk inhibitor p21 through an E2F mechanism the consequences of which are not to growth arrest cells but possibly to stabilize cyclin D1/cdk function.     

Reversal left diastolic ventriculo-atrial flow induced by asynchronism in a patient with dilated cardiopathy and VVI pacemaker]

Current therapy for glioma is suboptimal. The transfer of apoptosis genes to tumors constitutes one of the most promising strategies for cancer gene therapy. We have previously shown that massive apoptosis occurs when wild-type p53 or E2F-1 expression is induced in glioma. However, the mechanism of action and the efficiency in inducing apoptosis of these two proteins are not similar. Adenovirus-mediated p53 gene transfer is ineffective in causing apoptosis in glioma cells that retain wild-type p53 genotype or overexpress the p21 protein. The p16/Rb/E2F pathway is the most frequent target of genetic alterations in gliomas, and therefore constitutes a suitable target for gene therapy strategies. However, the transfer of either the p16 or Rb gene to glioma cells results in cytostatic effect. The E2F-1 protein is able to induce generalized apoptosis in gliomas independently of the p53, p16 or Rb status. In addition, p21- or p16-mediated growth arrest did not protect glioma cells from E2F-1-mediated apoptosis. The apoptotic molecule bax is induced in p53-mediated apoptosis, but bax is not induced in E2F-1-mediated apoptosis in glioma cells. Careful selection of patients may be necessary before designing therapeutic strategies using either p53 or E2F-1 as a therapeutic tools for glioma patients.     

Immunobiological approach to spinocerebellar degeneration--anti-Purkinje cell antibodies in paraneoplastic cerebellar degeneration

We analyzed the antibodies against cerebellum in five patients with paraneoplastic cerebellar degeneration (PCD). Four patients were found to have autoantibodies against rat brain, first one to cerebral 250 kd and 110 kd and cerebellar 110 kd proteins, second one to cerebral and cerebellar 98 kd and 68 kd proteins, third one to cerebellar 58 kd protein and fourth one to 58 kd protein especially in the cytoplasm and nucleus of Purkinje cells. We isolated a cDNA clone from a human cerebellar library to identify the target antigen for fourth antibody. Homology searches revealed a similarity with the zinc finger proteins. Zinc finger proteins are considered to regulate gene expression. Therefore, degeneration of this protein by its antibody may affect the synthesis of proteins in Purkinje cell and may cause cerebellar degeneration.