Erwin Lauwers

Characterization of lentiviral vector-mediated gene transfer in adult mouse brain

Lentiviral vectors are promising tools for gene transfer into the central nervous system. We have characterized in detail transduction with human immunodeficiency virus type 1 (HIV-1)-derived vectors encoding enhanced green fluorescent protein (eGFP) in the adult mouse brain. Different brain regions such as the striatum, hippocampus, and the lateral ventricle were targeted. The eGFP protein was transported anterogradely in the nigrostriatal pathway, but we have found no evidence of transport of the lentiviral vector particle. The performance levels of the different generations of packaging and transfer plasmid were compared. Omission of the accessory genes from the packaging plasmid resulted in a modest decrease in transgene expression. Inclusion of the woodchuck hepatitis posttranscriptional regulatory element, on the one hand, and the central polypurine tract and termination sequences, on the other hand, in the transfer vector each resulted in a 4- to 5-fold increase in transgene expression levels. Combination of both elements enhanced expression levels more than the sum of the individual components, suggesting a synergistic effect. In the serum of mice injected with lentiviral vectors a humoral response to vector proteins was detected, but this did not compromise transgene expression. Immune response to the transgene was found only in a minority of the animals.

Neuropathology and neurodegeneration in rodent brain induced by lentiviral vector-mediated overexpression of alpha-synuclein.

Two mutations in α‐synuclein, the main constituent of Lewy bodies, have been identified in familial Parkinsons disease. We have stereotactically injected lentiviral vectors encoding wild‐type and A30P mutant human α‐synuclein in different brain regions (striatum, substantia nigra, amygdala) of mice. Overexpression of α‐synuclein induced timedependent neuropathological changes reminiscent of Lewy pathology: abnormal accumulation of α‐synuclein in cell bodies and neurites, α‐synuclein‐positive neuritic varicosities and cytoplasmic inclusions that stained with ubiquitin antibodies and became larger and more frequent with time. After one year, α‐synuclein‐ and ubiquitin‐positive neurons displayed a degenerative morphology and a significant loss of α‐synuclein‐positive cells was observed. Similar findings were observed with both the wild‐type and the A30P mutant form of α‐synuclein and this in different brain regions. This indicates that overexpression of α‐synuclein is sufficient to induce Lewy‐like pathology and neurodegeneration and that this effect is not restricted to dopaminergic cells. Our data also demonstrate the use of lentiviral vectors to create animal models for neurodegenerative diseases.

Non-invasive imaging of neuropathology in a rat model of α-synuclein overexpression

Parkinsons disease is a neurodegenerative disorder affecting the dopaminergic neurons in the substantia nigra. Aggregation of alpha-synuclein appears to play a central role in the pathogenesis. Novel animal models for neurodegeneration have been generated by lentiviral vector-mediated locoregional overexpression of disease-associated genes in the adult brain. We have used lentiviral vectors to overexpress a clinical mutant of alpha-synuclein, A30P, in the rat substantia nigra. This overexpression induced time-dependent cytoplasmic and neuritic accumulation of alpha-synuclein and neurodegeneration. A subgroup of the rats developed asymmetric rotational behavior after administration of amphetamine. In addition, these animals displayed reduced dopamine transporter binding visualized by 123I-FP-CIT microSPECT imaging. The behavioral and microSPECT data were validated by histological analysis. There was a strong correlation between the reduction of dopaminergic neurons in the substantia nigra and the reduction of dopamine transporter binding in the striatum. MicroSPECT imaging enables non-invasive imaging of the neurodegeneration allowing longitudinal follow-up in this new animal model for Parkinsons disease and the evaluation of neuroprotective drugs.

In vivo type 1 cannabinoid receptor mapping in the 6-hydroxydopamine lesion rat model of Parkinson's disease

Type 1 cannabinoid (CB1) receptors are expressed in high concentrations in the central nervous system, including the basal ganglia, and could have direct or indirect effects on motor behavior through modulation of dopaminergic, glutamatergic and GABA-ergic neurotransmission. Using the CB1 receptor radioligand [(18)F]MK-9470 and small-animal PET, we investigated for the first time in vivo cerebral changes in [(18)F]MK-9470 binding in the 6-hydroxydopamine (6-OHDA) rat model of Parkinsons disease (PD), parallel to dopamine transporter (DAT) imaging, tyrosine hydroxylase (TH) staining, and behavioral measurements. In the 6-OHDA model, relative [(18)F]MK-9470 PET binding decreased in the contralateral cerebellum (-9%, p<0.0004) and caudate-putamen bilaterally (ipsilateral -8%, contralateral -7%; p=0.001 and p<0.0003, respectively). The number of TH(+) neurons in the substantia nigra was inversely correlated to CB1 receptor binding in the ipsilateral cerebellum (p=1.10(-6)). The behavioral outcome was positively related to regional CB1 receptor binding in the contralateral somatosensory cortex (p=4.10(-6)). In vivo [(18)F]MK-9470 PET imaging points to changes in endocannabinoid transmission, specifically for CB1 receptors in the 6-OHDA model of PD, with mainly involvement of the caudate-putamen, but also distant regions of the motor circuitry, including the cerebellum and somatosensory cortex.

Metabolic–dopaminergic mapping of the 6-hydroxydopamine rat model for Parkinson’s disease

PurposeThe unilateral 6-hydroxydopamine (6-OHDA) lesion rat model is a well-known acute model for Parkinson’s disease (PD). Its validity has been supported by invasive histology, behavioral studies and electrophysiology. Here, we have characterized this model in vivo by multitracer imaging [glucose metabolism and dopamine transporter (DAT)] in relation to behavioral and histological parameters.MethodsEighteen female adult Wistar rats (eight 6-OHDA-lesioned, ten controls) were investigated using multitracer [18F]-fluoro-2-deoxy-D-glucose (FDG) and [18F]-FECT {2′-[18F]-fluoroethyl-(1R-2-exo-3-exe)-8-methyl-3-(4-chlorophenyl)-8-azabicyclo(3.2.1)-octane-2-carboxylate} small animal positron emission tomography (PET). Relative glucose metabolism and parametric DAT binding images were anatomically standardized to Paxinos space and analyzed on a voxel-basis using SPM2, supplemented by a template-based predefined volumes-of-interest approach. Behavior was characterized by the limb-use asymmetry test; dopaminergic innervation was validated by in vitro tyrosine hydroxylase staining.ResultsIn the 6-OHDA model, significant glucose hypometabolism is present in the ipsilateral sensory-motor cortex (−6.3%; p = 4 × 10−6). DAT binding was severely decreased in the ipsilateral caudate-putamen, nucleus accumbens and substantia nigra (all p < 5 × 10−9), as confirmed by the behavioral and histological outcomes. Correlation analysis revealed a positive relationship between the degree of DAT impairment and the change in glucose metabolism in the ipsilateral hippocampus (p = 3 × 10−5), while cerebellar glucose metabolism was inversely correlated to the level of DAT impairment (p < 3 × 10−4).ConclusionsIn vivo cerebral mapping of 6-OHDA-lesioned rats using [18F]-FDG and [18F]-FECT small animal PET shows molecular–functional correspondence to the cortico-subcortical network impairments observed in PD patients. This provides a further molecular validation supporting the validity of the 6-OHDA lesion model to mimic multiple aspects of human PD.