45 research outputs found
CAV-2 Vector Development and Gene Transfer in the Central and Peripheral Nervous Systems
The options available for genetic modification of cells of the central nervous system (CNS) have greatly increased in the last decade. The current panoply of viral and nonviral vectors provides multifunctional platforms to deliver expression cassettes to many structures and nuclei. These cassettes can replace defective genes, modify a given pathway perturbed by diseases, or express proteins that can be selectively activated by drugs or light to extinguish or excite neurons. This review focuses on the use of canine adenovirus type 2 (CAV-2) vectors for gene transfer to neurons in the brain, spinal cord, and peripheral nervous system. We discuss (1) recent advances in vector production, (2) why CAV-2 vectors preferentially transduce neurons, (3) the mechanism underlying their widespread distribution via retrograde axonal transport, (4) how CAV-2 vectors have been used to address structure/function, and (5) their therapeutic applications
The Coexpression of Reelin and Neuronal Nitric Oxide Synthase in a Subpopulation of Dentate Gyrus Neurons Is Downregulated in Heterozygous Reeler Mice
Reelin is an extracellular matrix protein expressed in several interneuron subtypes in the hippocampus and dentate gyrus. Neuronal nitric oxide synthase (nNOS) is also expressed by interneurons in these areas. We investigated whether reelin and nNOS are co-localized in the same population of hippocampal interneurons, and whether this colocalization is altered in the heterozygous reeler mouse. We found colocalization of nNOS in reelin-positive cells in the CA1 stratum radiatum and lacunosum moleculare, the CA3 stratum radiatum, and the dentate gyrus subgranular zone, molecular layer, and hilus. In heterozygous reeler mice, the colocalization of nNOS in reelin-positive cells was significantly decreased only in the subgranular zone and molecular layer. The coexpression of reelin and nNOS in several hippocampal regions suggests that reelin and nNOS may work synergistically to promote glutamatergic function, and the loss of this coexpression in heterozygous reeler mice may underlie some of the behavioral deficits observed in these animals
Epithelial de-differentiation triggered by co-ordinate epigenetic inactivation of the EHF and CDX1 transcription factors drives colorectal cancer progression
Epigenetics; Tumour-suppressor proteinsEpigenética; Proteínas supresoras de tumoresEpigenètica; Proteïnes supresores de tumorsColorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC.This project was supported by NHMRC project grant (1107831), a Cancer Council Victoria Grant (1164674) and the Operational Infrastructure Support Programme, Victorian Government, Australia. JMM was supported by a NHMRC Senior Research Fellowship (1046092). IYL was supported by F J Fletcher Research Scholarship and Randal and Louisa Alcock Scholarship from the University of Melbourne. LJJ was supported by La Trobe University Australian Postgraduate Awards. IN was supported by La Trobe University Postgraduate Research Scholarship. JWTT was supported by the University of Melbourne Australian Postgraduate Awards. OMS is a National Health and Medical Research Council (NHMRC) Senior Research Fellow (APP1136119). Open Access funding enabled and organized by CAUL and its Member Institutions
Detection of cannabinoid receptors CB1 and CB2 within basal ganglia output neurons in macaques: changes following experimental parkinsonism
Abstract Although type 1 cannabinoid receptors (CB1-
Rs) are expressed abundantly throughout the brain, the
presence of type 2 cannabinoid receptors (CB2Rs) in neurons
is still somewhat controversial. Taking advantage of
newly designed CB1R and CB2R mRNA riboprobes, we
demonstrate by PCR and in situ hybridization that transcripts
for both cannabinoid receptors are present within
labeled pallidothalamic-projecting neurons of control and
MPTP-treated macaques, whereas the expression is markedly
reduced in dyskinetic animals. Moreover, an in situ
proximity ligation assay was used to qualitatively assess
the presence of CB1Rs and CB2Rs, as well as CB1R–CB2R
heteromers within basal ganglia output neurons in all
animal groups (control, parkinsonian and dyskinetic
macaques). A marked reduction in the number of CB1Rs,
CB2Rs and CB1R–CB2R heteromers was found in dyskinetic
animals, mimicking the observed reduction in CB1R
and CB2R mRNA expression levels. The fact that chronic
levodopa treatment disrupted CB1R–CB2R heteromeric
complexes should be taken into consideration when
designing new drugs acting on cannabinoid receptor
heteromers
Glucocerebrosidase expression patterns in the non-human primate brain
Glucocerebrosidase (GCase) is a lysosomal
enzyme encoded by the GBA1 gene. Mutations in GBA1
gene lead to Gaucher’s disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase
activity, therefore promoting the aggregation of alphasynuclein, a common neuropathological finding underlying
Parkinson’s disease (PD) and dementia with Lewy bodies.
However, it is also worth noting that a direct link between
GBA1 mutations and alpha-synuclein aggregation indicating cause and effect is still lacking, with limited experimental evidence to date. Bearing in mind that a number of
strategies increasing GCase expression for the treatment of
PD are currently under development, here we sought to
analyze the baseline expression of GCase in the brain of
Macaca fascicularis, which has often been considered as
the gold-standard animal model of PD. Although as with
other lysosomal enzymes, GCase is expected to be ubiquitously expressed, here a number of regional variations
have been consistently found, together with several specific
neurochemical phenotypes expressing very high levels of
GCase. In this regard, the most enriched expression of
GCase was constantly found in cholinergic neurons from
the nucleus basalis of Meynert, dopaminergic cells in the
substantia nigra pars compacta, serotoninergic neurons
from the raphe nuclei, as well as in noradrenergic neurons
located in the locus ceruleus. Moreover, it is also worth
noting that moderate levels of expression were also found
in a number of areas within the paleocortex and archicortex, such as the entorhinal cortex and the hippocampal
formation, respectively
Calbindin content and differential vulnerability of midbrain efferent dopaminergic neurons in macaques
Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from
degeneration. Although a direct link between CB content and differential vulnerability of
dopaminergic neurons has long been accepted, factors other than CB have also been
suggested, particularly those related to the dopamine transporter. Indeed, several studies
have reported that CB levels are not causally related to the differential vulnerability
of dopaminergic neurons against neurotoxins. Here we have used dual stains for
tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize
dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral
tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB.
In control animals, the highest percentages of co-localization were found in VTA (58.2%),
followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB
expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA
was similar to control monkeys (62.1%), whereas most of the few surviving neurons in
the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within
identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For
this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate
nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen,
whereas two more monkeys were injected with CTB into the internal division of the globus
pallidus (GPi). As expected, all the nigrocaudate- and nigroputamen-projecting neurons
were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative
for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and
CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTPinduced
degeneration, our data clearly demonstrated that these neurons are not giving rise
to nigrostriatal projections and indeed CB-ir/TH-ir neurons only originate nigroextrastriatal
projections
Adeno-associated viral vectors serotype 8 for cell-specific delivery of therapeutic genes in the central nervous system
Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters