10 research outputs found
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 Gaucherâs disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase
activity, therefore promoting the aggregation of alphasynuclein, a common neuropathological finding underlying
Parkinsonâs 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 ÎČ2 (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
Glucocerebrosidase gene therapy in animal models of Parkinson Disease
ParkinsonÂŽs Disease (PD) is the first most common movement disorder and second most common neurodegenerative disease that affects 1.5% of the global population above 65 years old. Although the incidence of PD varies depending on genetic, race/ethnicity and environmental factors, advanced age has been appointed as the main risk factor for PD
Glucocerebrosidase gene therapy in animal models of Parkinson Disease
ParkinsonÂŽs Disease (PD) is the first most common movement disorder and second most common neurodegenerative disease that affects 1.5% of the global population above 65 years old. Although the incidence of PD varies depending on genetic, race/ethnicity and environmental factors, advanced age has been appointed as the main risk factor for PD
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 Gaucherâs disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase
activity, therefore promoting the aggregation of alphasynuclein, a common neuropathological finding underlying
Parkinsonâs 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 ÎČ2 (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