Imaging brain amyloid deposition using grating-based differential phase contrast tomography.

One of the core pathological features of Alzheimer's disease (AD) is the accumulation of amyloid plaques in the brain. Current efforts of medical imaging research aim at visualizing amyloid plaques in living patients in order to evaluate the progression of the pathology, but also to facilitate the diagnosis of AD at the prodromal stage. In this study, we evaluated the capabilities of a new experimental imaging setup to image amyloid plaques in the brain of a transgenic mouse model of Alzheimer's disease. This imaging setup relies on a grating interferometer at a synchrotron X-ray source to measure the differential phase contrast between brain tissue and amyloid plaques. It provides high-resolution images with a large field of view, making it possible to scan an entire mouse brain. Here, we showed that this setup yields sufficient contrast to detect amyloid plaques and to quantify automatically several important structural parameters, such as their size and their regional density in 3D, on the scale of a whole mouse brain. Whilst future developments are required to apply this technique in vivo, this grating-based setup already gives the possibility to perform powerful studies aiming at quantifying the amyloid pathology in mouse models of AD and might accelerate the evaluation of anti-amyloid compounds. In addition, this technique may also facilitate the development of other amyloid imaging methods such as positron emission tomography (PET) by providing convenient high-resolution 3D data of the plaque distribution for multimodal comparison.

In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice.

Metachromatic leukodystrophy (MLD) is a lipidosis caused by deficiency of arylsulfatase A (ARSA). Although the genetics of MLD are known, its pathophysiology is not understood. The disease leads to progressive demyelination and early death and no effective treatment is available. We used lentiviral vectors to deliver a functional ARSA gene (human ARSA) into the brain of adult mice with germ-line inactivation of the mouse gene encoding ARSA, As2. We report sustained expression of active enzyme throughout a large portion of the brain, with long-term protection from development of neuropathology and hippocampal-related learning impairments. We show that selective degeneration of hippocampal neurons is a central step in disease pathogenesis, and provide evidence that in vivo transfer of ARSA by lentiviral vectors reverts the disease phenotype in all investigated areas. Therefore, in vivo gene therapy offers a unique option for MLD and other storage diseases affecting the central nervous system.

Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinson's disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

Rab1A over-expression prevents Golgi apparatus fragmentation and partially corrects motor deficits in an alpha-synuclein based rat model of Parkinson's disease.

Although the overabundance of human alpha-synuclein in nigral dopaminergic neurons is considered to play a pathogenic role in Parkinson's disease (PD), it remains unclear how alpha-synuclein leads to neuronal degeneration and motor symptoms. Here, we explored the effect of human alpha-synuclein in the rat substantia nigra following AAV-mediated gene delivery inducing a moderate loss of dopaminergic neurons together with motor impairments. A significant fraction of the surviving nigral neurons were found to express human αSyn and displayed a pathological fragmentation of the Golgi apparatus. This observation prompted further investigation on the role of the secretory pathway, in particular at the ER/Golgi level, in alpha-synuclein toxicity. To address this question, we co-expressed human alpha-synuclein with Rab1A, a regulator of ER-to-Golgi vesicular trafficking, and found a significant reduction of Golgi fragmentation. Rab1A did not protect the dopaminergic neurons from the alpha-synuclein-induced degeneration that occurred within several months following vector injection. However, we observed in animals co-expressing Rab1A an improvement of motor behavior that correlates with the rescue of normal Golgi morphology in alpha-synuclein-expressing dopaminergic neurons. The non-prenylable mutant Rab1A-DeltaCC did not produce any of the effects observed with the wild-type form of Rab1A, linking the protective role of Rab1A with its activity in ER-to-Golgi vesicular trafficking. In conclusion, Rab1A can rescue the Golgi fragmentation caused by the overabundance of alpha-synuclein in nigral dopaminergic neurons, improving the ability of the surviving neurons to control motor function in hemiparkinsonian animals.

Attenuation of 6-OHDA-induced neurotoxicity in glutathione peroxidase transgenic mice.

Normal cellular metabolism produces oxidants which are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidants and antioxidants has been postulated to lead to the degeneration of specific populations of neurons in neurodegenerative diseases, e.g. Parkinson's disease. The present study investigates whether overexpression of glutathione peroxidase, the enzyme which metabolizes hydrogen peroxide to water, can prevent or slow down neuronal injury in an animal model of Parkinson's disease. Transgenic mice overexpressing the human glutathione peroxidase gene under the control of the mouse hydroxymethylglutaryl-coenzyme A promoter and genetically matched control mice were injected intracerebroventricularly with the dopaminergic neurotoxin 6-hydroxydopamine. Seven days after injection, the number of tyrosine hydroxylase-positive nigral dopaminergic neurons was decreased by 52.4% and 20.5% in 6-hydroxydopamine-injected control and glutathione peroxidase transgenic mice, respectively. Similarly, 3 days after injection of the neurotoxin, striatal dopamine was decreased by 71.2% and 56.5%, respectively. Overexpression of glutathione peroxidase therefore partially protects dopaminergic neurons against 6-hydroxydopamine-induced toxicity.

Neuroprotective effect of interleukin-6 and IL6/IL6R chimera in the quinolinic acid rat model of Huntington's syndrome.

Ciliary neurotrophic factor prevents behavioural deficits and striatal degeneration in rat and primate models of Huntington's disease. Interleukin-6, another member of the cytokine family, and the chimeric molecule (IL6/IL6R) in which interleukin-6 and its soluble receptor are fused, have been shown to exert trophic action on various neuronal populations in the central nervous system. Therefore, we investigated the neuroprotective effect of these two molecules in the quinolinic acid model of Huntington's disease. LacZ-, interleukin-6- and IL6/IL6R-expressing lentiviral vectors were stereotaxically injected into the striatum of Wistar rats. Three weeks later the animals were lesioned through the intrastriatal injection of 180 nmol of quinolinic acid. The extent of the striatal damage was significantly diminished in the rats that had been treated with interleukin-6 or IL6/IL6R. The neuroprotective effect was, however, more pronounced with the IL6/IL6R chimera than with interleukin-6 as indicated by the volume of the lesions (38.6 +/- 10% in the IL6/IL6R group, 63.3 +/- 3.6% in the IL-6 group and 84.3 +/-2.9% in the control group). Quantitative analysis of striatal interneurons further demonstrated that the IL6/IL6R chimera is more neuroprotective than IL-6 on ChAT- and NADPH-d-immunoreactive neurons. These results suggest that the IL6/IL6R chimera is a potential treatment for Huntington's disease.

Lentiviral vectors as a gene delivery system in the mouse midbrain: cellular and behavioral improvements in a 6-OHDA model of Parkinson's disease using GDNF.

Local delivery of therapeutic molecules represents one of the limiting factors for the treatment of neurodegenerative disorders. In vivo gene transfer using viral vectors constitutes a powerful strategy to overcome this limitation. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse midbrain in the perspective of screening biotherapeutic molecules in mouse models of Parkinson's disease. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect approximately 40,000 cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinson's disease. Lentiviral vectors carrying the cDNA for GDNF or mutated GDNF were unilaterally injected above the substantia nigra of C57BL/6j mice. Two weeks later, the animals were lesioned ipsilaterally with 6-hydroxydopamine into the striatum. Apomorphine-induced rotation was significantly decreased in the GDNF-injected group compared to control animals. Moreover, GDNF efficiently protected 69.5% of the tyrosine hydroxylase-positive cells in the substantia nigra against 6-hydroxydopamine-induced toxicity compared to 33.1% with control mutated GDNF. These data indicate that lentiviral vectors constitute a powerful gene delivery system for the screening of therapeutic molecules in mouse models of Parkinson's disease.