ORMOSIL nanoparticles as a non-viral gene delivery vector for modeling polyglutamine induced brain pathology.

Studies have shown the presence of expanded polyQ containing proteins in brain cells related to Huntington disease (HD) and other poly-glutamine disorders. We report the use of organically modified silica (ORMOSIL) nanoparticles as an efficient non-viral gene carrier in an effort to model brain pathology associated with those disorders induced by expanded polyQ peptides. In experiment 1, plasmids expressing Hemaglutinin-tagged polypeptides with 20 glutamine repeats (Q20) or with extended 127-glutamine repeats (Q127) were complexed with ORMOSIL nanoparticles and injected twice (2 weeks apart) into the lateral ventricle of the mouse brain. Fourteen days post-injection of Q127, immunocytochemistry revealed the presence of the characteristic nuclear and cytoplasmic Q127 aggregates in numerous striatal, septal and neocortical neuronal cells as well as ubiquitin-containing aggregates indicative of the neuronal pathology. The mice receiving Q127 showed a marked increase in the reactive GFAP (+) astrocytes in striatum, septum and brain cortex, further indicating the neurodegenerative changes, accompanied by motor impairments. In experiment 2, plasmids Q20 or Q127 were complexed with ORMOSIL and were injected into the brain lateral ventricle or directly into the striatum of adult rats. In both routes of transfection, Q127 induced the appearance of reactive GFAP (+) astrocytes and activated ED1 antigen expressing microglia. An increase in the size of the lateral ventricle was also observed in rats receiving Q127. In transgenic mouse polyQ models, extensive pathologies occur outside the nervous system and the observed brain pathologies could reflect developmental effects of the toxic polyQ proteins. Our experiments show that the nervous tissue restricted expression of poly Q-extended peptides in adult brain is sufficient to evoke neuropathologies associated with HD and other polyQ disorders. Thus, nanotechnology can be employed to model pathological and behavioral aspects of genetic brain diseases in mice as well as in other species, providing a novel research tool for in vivo testing of single or multi-gene therapies.

Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina.

BACKGROUND: Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI) can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli beta-galactosidase or enhanced green fluorescence protein (EGFP) using PEI. RESULTS: Optimal transfection efficiency was observed with 1 microg/ml of plasmid DNA and 5 microg/ml PEI. Expression of beta-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with approximately 9% of the neurons expressing beta-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector. CONCLUSIONS: These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures.

Immunodetection of Parkin protein in vertebrate and invertebrate brains: a comparative study using specific antibodies.

Parkin is an intracellular protein that plays a significant role in the etiopathogenesis of autosomal recessive juvenile parkinsonism. Using immunoblot methods, we found Parkin isoforms varying from 54 to 58 kDa in rat, mouse, bird, frog and fruit-fly brains. Immunocytochemical studies carried out in rats, mice and birds demonstrated multiple cell types bearing the phenotype for Parkin throughout telencephalic, diencephalic, mesencephalic and metencephalic brain structures. While in some instances Parkin-containing neurons tended to be grouped into clusters, the majority of these labeled nerve cells were widely scattered throughout the neuraxis. The topographical distribution and organizational pattern of Parkin within major functional brain circuits was comparable in both rats and mice. However, the subcellular localization of Parkin was found to vary significantly as a function of antibody reactivity. A consistent cytoplasmic labeling for Parkin was observed in rodent tissue incubated with a polyclonal antibody raised against the human Parkin protein and having an identical amino-acid sequence with that of the rat. In contrast, rodent tissue alternately incubated with a polyclonal antibody raised against a different region of the same human Parkin protein but having 10 mismatched amino-acid sequence changes with those of the rat and mouse, resulted in nuclear labeling for Parkin in rat but not mouse neurons. This difference in epitope recognition, however, was reversed when mouse brain tissue was heated at 80 degrees C, apparently unmasking target epitopes against which the antisera were directed. Collectively, these results show a high degree of conservation in the cellular identity of Parkin in animals as different as drosophilids and mammals and points to the possibility that the biochemical specificities of Parkin, including analogous functional roles, may have been conserved during the course of evolution.