p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53ß are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53ß, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53ß overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53ß expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.

Management and outcome of CSF-JC virus PCR-negative PML in a natalizumab-treated patient with MS.

OBJECTIVE: To describe the diagnosis and management of a 49-year-old woman with multiple sclerosis (MS) developing a progressive hemiparesis and expanding MRI lesion suspicious of progressive multifocal leukoencephalopathy (PML) 19 months after starting natalizumab. RESULTS: Polyomavirus JC (JCV)-specific qPCR in CSF was repeatedly negative, but JCV-specific antibodies indicated intrathecal production. Brain biopsy tissue taken 17 weeks after natalizumab discontinuation and plasmapheresis was positive for JCV DNA with characteristic rearrangements of the noncoding control region, but histology and immunohistochemistry were not informative except for pathologic features compatible with immune reconstitution inflammatory syndrome. A total of 22 months later, the clinical status had returned close to baseline level paralleled by marked improvement of neuroradiologic abnormalities. CONCLUSIONS: This case illustrates diagnostic challenges in the context of incomplete suppression of immune surveillance and the potential of recovery of PML associated with efficient immune function restitution.

Immune reconstitution inflammatory syndrome in natalizumab-associated PML.

OBJECTIVE: To study the outcome of patients with multiple sclerosis (MS) and with natalizumab-associated progressive multifocal leukoencephalopathy (PML) and immune reconstitution inflammatory syndrome (IRIS). METHODS: MedWatch reports from Biogen-Idec (manufacturer of natalizumab, Tysabri(®)) were reviewed which comprised all 42 cases of natalizumab-related PML cases since its reintroduction until March 2010. RESULTS: All except 2 patients with natalizumab-related PML were managed by discontinuation of natalizumab and plasmapheresis/immunoadsorption (PLEX/IA). Seventeen patients had contrast enhancement of PML lesions on neuroimaging at the time of diagnosis before withdrawal/removal of natalizumab (early-PML-IRIS) and 23 patients developed contrast enhancement only after withdrawal/removal of natalizumab (late-PML-IRIS). All patients developed IRIS. IRIS was defined as worsening of neurologic deficits during the immune reconstitution following discontinuation of natalizumab, corroborated by inflammatory changes on neuroimaging. Following PLEX/IA, JC viral load in CSF increased by >10 fold in those with early-PML-IRIS but <2 fold in late-PML-IRIS. IRIS developed earlier and was more severe in early-PML-IRIS (p < 0.05). At the last follow-up, all patients had worse EDSS scores but this was higher in patients with early-PML-IRIS compared to those with late-PML-IRIS (p > 0.05). Mortality was comparable between the 2 groups, 29.4 ± 11% vs 21.7 ± 8.8%. Corticosteroid therapy during IRIS was associated with better Expanded Disability Status Scale outcome, p < 0.05. CONCLUSION: Early immunologic rebound in natalizumab-associated PML has worse survival and neurologic outcome. PLEX/IA may accelerate IRIS and its impact on the final outcome is unclear. Corticosteroid therapy provides a modest benefit and needs to be systemically studied in a controlled manner in the management of natalizumab-associated PML-IRIS.

Differences in NMDA receptor expression during human development determine the response of neurons to HIV-tat-mediated neurotoxicity.

HIV infection of the CNS can result in neurologic dysfunction in a significant number of infected individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV infection in neurons. Thus, neuronal damage and dropout are likely due to indirect effects of HIV infection of other CNS cells, through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivating protein tat. We and others demonstrated that tat induces apoptosis in differentiated mature human neurons. We now demonstrate that the high level of tat toxicity observed in human neurons involves specific developmental stages that correlate with N-methyl-D-aspartate receptor (NMDAR) expression, and that tat toxicity is also dependent upon the species being analyzed. Our results indicate that tat treatment of primary cultures of differentiated human neurons with significant amounts of NMDAR expression induces extensive apoptosis. In contrast, tat treatment induces only low levels of apoptosis in primary cultures of immature human neurons with low or minimal expression of NMDAR. In addition, tat treatment has minimal effect on rat hippocampal neurons in culture, despite their high expression of NMDAR. We propose that this difference may be due to low expression of the NR2A subunit. These findings are important for an understanding of the many differences among tissue culture systems and species used to study HIV-tat-mediated toxicity.

Human brain derived cell culture models of HIV-1 infection.

During the clinical course of acquired immune deficiency syndrome, infection of the CNS by human immunodeficiency virus-1 (HIV-1) may ultimately result in the impairment of cognitive, behavioral and motor functions. Viral neuropathogenesis involves inflammatory molecules and neurotoxins produced from infected and immune-activated lymphocytes, microglial cells and astrocytes. Here, we discuss the current understanding of HIV-1 infection of the CNS and various cell culture systems from the developing human brain in order to study the neurobiology of HIV-1 infection, the mechanisms contributing to HIV-1 infection, and disease progression.

Establishment of a new human immortalized chondrocyte cell line.

Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorescence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium and seeding on a hyaluronan-based three-dimensional biomaterial. Immortalized cells were able to re-express the main markers of chondrocyte phenotype, both at mRNA and protein levels, under the two defined cultured conditions used. The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation, and of the etiopathogenesis of many rheumatic diseases.

Human articular chondrocytes immortalized by HPV-16 E6 and E7 genes: Maintenance of differentiated phenotype under defined culture conditions.

OBJECTIVE: To establish an immortalized normal human articular chondrocyte line which could be useful for a better understanding of cell molecular mechanisms relevant for the development of new therapeutic approaches in rheumatic diseases. DESIGN: Chondrocytes from human adult articular healthy cartilage were transfected in primary culture with a plasmid containing two human papilloma virus type 16 (HPV-16) early function genes: E6 and E7, using the highly efficient cationic liposome-mediated (lipofection) procedure. The transfection was verified by reverse transcriptase-polymerase chain reaction analysis of E7 mRNA and by immunofluorence localization of the E7 protein in the cell cytoplasm. The established chondrocyte cell line was examined in monolayer and in two culture conditions that were described to re-induce differentiated characteristics: culturing in a serum-free defined medium supplemented with an insulin-containing serum substitute and seeding on a hyaluronan-based non-woven structured biomaterial. The expression of markers characteristic of cartilage was shown in the mRNA by reverse transcriptase-polymerase chain reaction. Immunohistological staining and Western blotting analysis were performed to evaluate type II collagen synthesis. Proteoglycans deposition was detected by Alcian Blue staining. A Field Emission In Lens Scanning Microscopy was used to look at the morphology of the immortalized cells at very high magnification. RESULTS: Normal human articular chondrocytes were efficiently transfected leading to the establishment of an immortalized cell line as confirmed by HPV-16 E7 mRNA and protein detection. These cells were able to re-express type II collagen both at mRNA and protein levels under the two defined cultured conditions we used, still maintaining type I collagen expression. Collagen IX mRNA was present only in early primary culture while collagen type X and aggrecan transcripts were always detected. Alcian Blue staining showed a proteoglycan-rich matrix production. The ultrastructural analysis of the immortalized cells revealed that their morphology strictly resembled that of normal chondrocytes. CONCLUSIONS: The cell line that we obtained may be a useful tool for increasing our knowledge of the genetic and biochemical events involved in the processes of cartilage growth and differentiation. Moreover, it appears to be a suitable model for pharmacological and toxicological studies related to rheumatic diseases relevant to humans.

JC virus multiplication in human hematopoietic progenitor cells requires the NF-1 class D transcription factor.

JCV, a small DNA virus of the polyomavirus family, has been shown to infect glial cells of the central nervous system, hematopoietic progenitor cells, and immune system lymphocytes. A family of DNA binding proteins called nuclear factor-1 (NF-1) has been linked with site-coding specific transcription of cellular and viral genes and replication of some viruses, including JC virus (JCV). It is unclear which NF-1 gene product must be expressed by cells to promote JCV multiplication. Previously, it was shown that elevated levels of NF-1 class D mRNA were expressed by human brain cells that are highly susceptible to JCV infection but not by JCV nonpermissive HeLa cells. Recently, we reported that CD34(+) precursor cells of the KG-1 line, when treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), differentiated to cells with macrophage-like characteristics and lost susceptibility to JCV infection. These studies have now been extended by asking whether loss of JCV susceptibility by PMA-treated KG-1 cells is linked with alterations in levels of NF-1 class D expression. Using reverse transcription-PCR, we have found that PMA-treated KG-1 cells express mRNA that codes for all four classes of NF-1 proteins, although different levels of RNA expression were observed in the hematopoietic cells differentiated into macrophages. Northern hybridization confirms that the expression of NF-1 class D gene is lower in JCV nonpermissive PMA-treated KG-1 cells compared with non-PMA-treated cells. Further, using gel mobility shift assays, we were able to show the induction of specific NF-1-DNA complexes in KG-1 cells undergoing PMA treatment. The binding increases in direct relation to the duration of PMA treatment. These results suggest that the binding pattern of NF-1 class members may change in hematopoietic precursor cells, such as KG-1, as they undergo differentiation to macrophage-like cells. Transfection of PMA-treated KG-1 cells with an NF-1 class D expression vector restored the susceptibility of these cells to JCV infection, while the transfection of PMA-treated KG-1 cells with NF-1 class A, B, and C vectors was not able to restore JCV susceptibility. These data collectively suggest that selective expression of NF-1 class D has a regulatory role in JCV multiplication.

In vitro generation and transplantation of precursor-derived human dopamine neurons.

The use of in vitro expanded human CNS precursors has the potential to overcome some of the ethical, logistic and technical problems of fetal tissue transplantation in Parkinson disease. Cultured rat mesencephalic precursors proliferate in response to bFGF and upon mitogen withdrawal, differentiate into functional dopamine neurons that alleviate motor symptoms in Parkinsonian rats (Studer et al. [1998] Nat. Neurosci. 1:290-295). The successful clinical application of CNS precursor technology in Parkinson disease will depend on the efficient in vitro generation of human dopaminergic neurons. We demonstrate that human dopamine neurons can be generated from both midbrain and cortical precursors. Transplantation of midbrain precursor-derived dopamine neurons into Parkinsonian rats resulted in grafts rich in tyrosine hydroxylase positive neurons 6 weeks after transplantation. No surviving tyrosine hydroxylase positive neurons could be detected when dopamine neurons derived from cortical precursors were grafted. Our data demonstrate in vitro derivation of human dopamine neurons from expanded CNS precursors and encourage further studies that systematically address in vivo function and clinical potential.

A classification scheme for human polyomavirus JCV variants based on the nucleotide sequence of the noncoding regulatory region.

The human polyomavirus JCV is responsible for the central nervous system (CNS) demyelination observed in cases of progressive multifocal leukoencephalopathy (PML). Lytic infection of oligodendrocytes, the cells that constitute the basis of myelin in the CNS, is established by JCV in conjunction with immunosuppressive conditions. Beyond this, however, many questions related to JCV pathogenesis remain unanswered. The JCV regulatory region is a hypervariable noncoding sequence positioned between the early and late protein-coding regions. The particular nucleotide sequence of a JCV regulatory region affects levels of viral transcription and replication. Modifications to this promoter/enhancer structure can alter the cellular host range and may be responsible for switching JCV between states of lytic and latent infection. The regulatory region structure has, therefore, been used to distinguish JCV variants. Nucleotide sequencing studies have uncovered numerous variations of regulatory region structure. Until now, however, no inclusive nomenclature existed that linked variants by regulatory region structure and/or activity. We have arranged all known variant JCV regulatory regions into quadrants according to the integration of particular sequence sections and repetition of sequence section groups. This arrangement of regulatory regions results in an updated nomenclature that is well-suited for describing the relationships between JCV variants. Four distinct structural forms (I-S, I-R, II-S, and II-R) are defined along with tissue tropisms. This design provides logical connections between the variant regulatory regions and may be useful for elucidating crucial steps in JCV pathogenesis.

Utility of human neural cells and cell lines for anti-viral vaccine research and development.

Specific cell types such as neurons, astrocytes, and microglial cells can be isolated from a mixed population of human foetal brain cells and cultured for extended periods. The rapid expansion of cell populations occurring during culture has accommodated the large-scale growth and production of various neurotropic viruses. Furthermore, neural cell lines derived from CNS tumours or by the immortalization of primary cells have also been established and used for studies of viral pathogenesis. The potential to generate and expand selected populations of neural-derived cells should provide a new and abundant substrate for the production of viruses in vaccine development.

Grafting genetically engineered cells into the striatum of nonhuman primates.

An emerging new technology based on genetic engineering of viral vectors that can insert genes into the cells of living organisms may play a significant role in treating disorders of the central nervous system (CNS). Most neurodegenerative disorders affect focal regions of the brain. Preventive and/ or palliative treatment strategies need to be targeted only to the diseased parts of the brain without affecting other regions. Administration of therapeutic genes specifically to the disease-affected regions of the brain may be more beneficial than current treatment strategies, which are largely based on systemically administering small molecules. The latter can result not only in peripheral side effects but also CNS side effects since the drugs can affect both targeted and nontargeted brain sites. In addition, many therapeutic agents are prevented from entering the brain by the blood-brain barrier (BBB). For these reasons, many otherwise potentially useful proteins, such as trophic factors, cannot be administered systemically (1).

Mechanisms of leukocyte trafficking into the CNS.

HIV-1 encephalitis occurs in up to one-third of HIV-1-infected individuals. The mechanisms through which this pathology develops are thought to involve viral passage across the blood-brain barrier (BBB), as well as entry of HIV-infected and/or uninfected inflammatory cells into the central nervous system (CNS). Viral proteins and cytokines may also contribute to the pathogenesis of encephalitis. We show that the chemokines SDF-1 and MCP-1 induce transmigration of uninfected human lymphocytes and monocytes across our model of the BBB, a co-culture of human fetal astrocytes and endothelial cells. We also demonstrate that the HIV-1 protein Tat induces adhesion molecule expression and chemokine production by human fetal astrocytes and microglia, which could further contribute to leukocyte entry into the CNS. Finally, our data indicate that inflammatory cytokines modulate the expression of CXCR4, a co-receptor for HIV-1, on human fetal astrocytes, suggesting that these cytokines may potentially modulate the infectability of astrocytes by HIV-1. These findings support the hypothesis that there may be several different mechanisms that contribute to the development and progression of HIV-1 encephalitis.

Stages of restricted HIV-1 infection in astrocyte cultures derived from human fetal brain tissue.

The predominant cell types infected by HIV-1 in AIDS associated encephalopathy are cells of the macrophage/microglial lineage. There has been consistent evidence, however, that astrocytes also become infected although not at the same frequency or level of multiplication as microglial cells. HIV-1 antigens and/or nucleic acid have been identified in astrocytes in brain autopsy tissue from both adult and pediatric AIDS cases. In cell cultures, HIV-1 infection of astrocytes results in an initial productive but non-cytopathogenic infection that diminishes to a viral persistence or latent state. Understanding the nature of HIV-1 infection of astrocytes, which represents the largest population of cells in the brain, will contribute to the understanding of AIDS encephalopathy and the dementia that occurs in nearly one-quarter of all AIDS patients.

Progressive multifocal leukoencephalopathy: JC virus induced demyelination in the immune compromised host.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system that predominantly affects immunocompromised individuals. The etiologic agent, JCV, is a widespread polyomavirus with a very specific target, the myelin-producing oligodendrocytes of the brain. During periods of immune suppression, the virus can be reactivated from lymphoid tissues and kidney, causing targeted myelin destruction and corresponding neurological deficits. The incidence of PML has increased in recent years, due in large part to the advent of AIDS and the growing number of immunodeficient individuals. Furthermore, previous serological studies have shown that greater than 80% of the human population has antibodies to JCV in circulation. When combined, these statistics highlight an increasing need to establish effective treatment regimens for infected individuals as well as strategies to identify those at risk for developing PML.