Insult-induced aberrant hippocampal neurogenesis: Functional consequences and possible therapeutic strategies.

Adult hippocampal neurogenesis plays a critical role in a wide spectrum of hippocampus-dependent functions. Brain pathologies that involve the hippocampus like epilepsy, stroke, and traumatic brain injury, are commonly associated with cognitive impairments and mood disorders. These insults can affect neural stem cells and the subsequent neurogenic cascade in the hippocampus, resulting in the induction of aberrant neurogenesis, which is thought to compromise hippocampal network function, thereby hampering hippocampus-dependent behavior. We here summarize recent preclinical literature on hippocampal insult-induced changes in neurogenesis and based on that, we propose that normalizing aberrant neurogenesis post-insult may help to prevent or rescue behavioral deficits which could help develop novel therapeutic strategies.

Inhibition of LRRK2 or Casein Kinase 1 Results in LRRK2 Protein Destabilization.

Mutations and variations in the leucine-rich repeat kinase 2 (LRRK2) gene are strongly associated with an increased risk to develop Parkinson's disease (PD). Most pathogenic LRRK2 mutations display increased kinase activity, which is believed to underlie LRRK2-mediated toxicity. Therefore, major efforts have been invested in the development of potent and selective LRRK2 kinase inhibitors. Several of these compounds have proven beneficial in cells and in vivo, even in a LRRK2 wild-type background. Therefore, LRRK2 kinase inhibition holds great promise as disease-modifying PD therapy, and is currently tested in preclinical and early clinical studies. One of the safety concerns is the development of lung pathology in mice and non-human primates, which is most likely related to the strongly reduced LRRK2 protein levels after LRRK2 kinase inhibition. In this study, we aimed to better understand the molecular consequences of chronic LRRK2 kinase inhibition, which may be pivotal in the further development of a LRRK2 kinase inhibitor-based PD therapy. We found that LRRK2 protein levels are not restored during long-term LRRK2 kinase inhibition, but are recovered upon inhibitor withdrawal. Interestingly, LRRK2 kinase inhibitor-induced destabilization does not occur in all pathogenic LRRK2 variants and the N-terminal part of LRRK2 appears to play a crucial role in this process. In addition, we identified CK1, an upstream kinase of LRRK2, as a regulator of LRRK2 protein stability in cell culture and in vivo. We propose that pharmacological LRRK2 kinase inhibition triggers a cascade that results in reduced CK1-mediated phosphorylation of yet unidentified LRRK2 phosphorylation sites. This process involves the N-terminus of LRRK2 and ultimately leads to LRRK2 protein degradation.

ɑ-Synuclein strains and seeding in Parkinson's disease, incidental Lewy body disease, dementia with Lewy bodies and multiple system atrophy: similarities and differences.

Several age-related neurodegenerative disorders are characterized by the deposition of aberrantly folded endogenous proteins. These proteins have prion-like propagation and amplification properties but so far appear nontransmissible between individuals. Because of the features they share with the prion protein, PrP, the characteristics of pathogenic protein aggregates in several progressive brain disorders, including different types of Lewy body diseases (LBDs), such as Parkinson's disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB), have been actively investigated. Even though the pleomorphic nature of these syndromes might suggest different underlying causes, ɑ-synuclein (ɑSyn) appears to play an important role in this heterogeneous group of diseases (the synucleinopathies). An attractive hypothesis is that different types of ɑSyn protein assemblies have a unique and causative role in distinct synucleinopathies. We will discuss the recent research progress on ɑSyn assemblies involved in PD, MSA and DLB; their behavior as strains; current spreading hypotheses; their ability to seed centrally and peripherally; and their implication for disease pathogenesis.

ATP13A2/PARK9 regulates endo-/lysosomal cargo sorting and proteostasis through a novel PI(3, 5)P2-mediated scaffolding function.

ATP13A2 (also called PARK9), is a transmembrane endo-/lysosomal-associated P5 type transport ATPase. Loss-of-function mutations in ATP13A2 result in the Kufor-Rakeb Syndrome (KRS), a form of autosomal Parkinson's disease (PD). In spite of a growing interest in ATP13A2, very little is known about its physiological role in stressed cells. Recent studies suggest that the N-terminal domain of ATP13A2 may hold key regulatory functions, but their nature remains incompletely understood. To this end, we generated a set of melanoma and neuroblastoma cell lines stably overexpressing wild-type (WT), catalytically inactive (D508N) and N-terminal mutants, or shRNA against ATP13A2. We found that under proteotoxic stress conditions, evoked by the proteasome inhibitor Bortezomib, endo-/lysosomal associated full-length ATP13A2 WT, catalytically-inactive or N-terminal fragment mutants, reduced the intracellular accumulation of ubiquitin-conjugated (Ub) proteins, independent of autophagic degradation. In contrast, ATP13A2 silencing increased the intracellular accumulation of Ub-proteins, a pattern also observed in patient-derived fibroblasts harbouring ATP13A2 loss-of function mutations. In treated cells, ATP13A2 evoked endocytic vesicle relocation and increased cargo export through nanovesicles. Expression of an ATP13A2 mutant abrogating PI(3,5)P2 binding or chemical inhibition of the PI(3,5)P2-generating enzyme PIKfyve, compromised vesicular trafficking/nanovesicles export and rescued intracellular accumulation of Ub-proteins in response to proteasomal inhibition. Hence, our study unravels a novel activity-independent scaffolding role of ATP13A2 in trafficking/export of intracellular cargo in response to proteotoxic stress.

Pharmacological LRRK2 kinase inhibition induces LRRK2 protein destabilization and proteasomal degradation.

Leucine-rich repeat kinase 2 (LRRK2) kinase activity is increased in several pathogenic mutations, including the most common mutation, G2019S, and is known to play a role in Parkinson's disease (PD) pathobiology. This has stimulated the development of potent, selective LRRK2 kinase inhibitors as one of the most prevailing disease-modifying therapeutic PD strategies. Although several lines of evidence support beneficial effects of LRRK2 kinase inhibitors, many questions need to be answered before clinical applications can be envisaged. Using six different LRRK2 kinase inhibitors, we show that LRRK2 kinase inhibition induces LRRK2 dephosphorylation and can reduce LRRK2 protein levels of overexpressed wild type and G2019S, but not A2016T or K1906M, LRRK2 as well as endogenous LRRK2 in mouse brain, lung and kidney. The inhibitor-induced reduction in LRRK2 levels could be reversed by proteasomal inhibition, but not by lysosomal inhibition, while mRNA levels remained unaffected. In addition, using LRRK2 S910A and S935A phosphorylation mutants, we show that dephosphorylation of these sites is not required for LRRK2 degradation. Increasing our insight in the molecular and cellular consequences of LRRK2 kinase inhibition will be crucial in the further development of LRRK2-based PD therapies.

α-Synuclein strains cause distinct synucleinopathies after local and systemic administration.

Misfolded protein aggregates represent a continuum with overlapping features in neurodegenerative diseases, but differences in protein components and affected brain regions. The molecular hallmark of synucleinopathies such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy are megadalton α-synuclein-rich deposits suggestive of one molecular event causing distinct disease phenotypes. Glial α-synuclein (α-SYN) filamentous deposits are prominent in multiple system atrophy and neuronal α-SYN inclusions are found in Parkinson's disease and dementia with Lewy bodies. The discovery of α-SYN assemblies with different structural characteristics or 'strains' has led to the hypothesis that strains could account for the different clinico-pathological traits within synucleinopathies. In this study we show that α-SYN strain conformation and seeding propensity lead to distinct histopathological and behavioural phenotypes. We assess the properties of structurally well-defined α-SYN assemblies (oligomers, ribbons and fibrils) after injection in rat brain. We prove that α-SYN strains amplify in vivo. Fibrils seem to be the major toxic strain, resulting in progressive motor impairment and cell death, whereas ribbons cause a distinct histopathological phenotype displaying Parkinson's disease and multiple system atrophy traits. Additionally, we show that α-SYN assemblies cross the blood-brain barrier and distribute to the central nervous system after intravenous injection. Our results demonstrate that distinct α-SYN strains display differential seeding capacities, inducing strain-specific pathology and neurotoxic phenotypes.

Depletion of RIPK3 or MLKL blocks TNF-driven necroptosis and switches towards a delayed RIPK1 kinase-dependent apoptosis.

In human cells, the RIPK1-RIPK3-MLKL-PGAM5-Drp1 axis drives tumor necrosis factor (TNF)-induced necroptosis through mitochondrial fission, but whether this pathway is conserved among mammals is not known. To answer this question, we analyzed the presence and functionality of the reported necroptotic axis in mice. As in humans, knockdown of receptor-interacting kinase-3 (RIPK3) or mixed lineage kinase domain like (MLKL) blocks TNF-induced necroptosis in L929 fibrosarcoma cells. However, repression of either of these proteins did not protect the cells from death, but instead induced a switch from TNF-induced necroptosis to receptor-interacting kinase-1 (RIPK1) kinase-dependent apoptosis. In addition, although mitochondrial fission also occurs during TNF-induced necroptosis in L929 cells, we found that knockdown of phosphoglycerate mutase 5 (PGAM5) and dynamin 1 like protein (Drp1) did not markedly protect the cells from TNF-induced necroptosis. Depletion of Pink1, a reported interactor of both PGAM5 and Drp1, did not affect TNF-induced necroptosis. These results indicate that in these murine cells mitochondrial fission and Pink1 dependent processes, including Pink-Parkin dependent mitophagy, apparently do not promote necroptosis. Our data demonstrate that the core components of the necrosome (RIPK1, RIPK3 and MLKL) are crucial to induce TNF-dependent necroptosis both in human and in mouse cells, but the associated mechanisms may differ between the two species or cell types.

Melatonin prevents dopaminergic cell loss induced by lentiviral vectors expressing A30P mutant alpha-synuclein.

Two hallmarks of Parkinson's disease (PD) are dopaminergic cell loss and the presence of cytoplasmic inclusions (Lewy bodies). Different point mutations in alpha-synuclein, the main constituent of Lewy bodies, have been identified in familial PD. Alpha-synuclein also constitutes one of the main components of Lewy bodies in sporadic cases of PD. Moreover, oxidant stress and generation of free radicals from both mitochondrial impairment and dopamine metabolism are considered to play critical roles in PD etiopathogenesis. Melatonin, a known potent antioxidant secreted by the pineal gland, may protect against the effect of several Parkinsonogenic compounds that are associated with progressive impairment of mitochondrial function and increased oxidative damage. However, the neuroprotective effect of melatonin has never been tested in the newly available genetic models of PD based on the viral expression of mutated alpha-synuclein. Lentiviral vectors encoding A30P mutant human alpha-synuclein (lenti-A30P) were stereotactically injected into the right substantia nigra of adult male Sprague-Dawley rats and neuroprotection was examined by administration of melatonin or vehicle from two days before nigral administration of lenti-A30P until eight weeks after injection. It was found that lenti-A30P induced a significant TH⁺ cell-loss both in the medial and lateral substantia nigra versus the contrallateral side injected with lenti-eGFP. However, melatonin administration showed a total neuroprotective effect in both regions of the substantia nigra. In conclusion, the data here show that melatonin is neuroprotective against mutant alpha-synuclein-induced injury in the substantia nigra.

Comparison of motor performance, brain biochemistry and histology of two A30P α-synuclein transgenic mouse strains.

Three point mutations in the SNCA gene encoding α-synuclein (aSyn) have been associated with autosomal dominant forms of Parkinson's disease. To better understand the role of the A30P mutant aSyn, we compared two transgenic mouse strains: a knock-in mouse with an introduced A30P point mutation in the wild-type (WT) gene (Snca(tm(A30P))) and a transgenic (Tg) mouse overexpressing the human A30P aSyn gene under the prion promoter [tg(Prnp-SNCA A30P)]. The brain aSyn load, motor performance, brain dopamine (DA) and sensitivity to 6-hydroxydopamine (6-OHDA) were studied in these mice. aSyn was evidently accumulating with age in all mice, particularly in tg(Prnp-SNCA A30P) Tg mice. There were no robust changes in basal locomotor activities of the mice of either line at 6 months, but after 1 year, tg(Prnp-SNCA A30P) Tg mice developed severe problems with vertical movements. However, the younger Tg mice had a reduced locomotor response to 1mg/kg of d-amphetamine. Snca(tm(A30P)) mice with the targeted mutation (Tm) were slightly hyperactive at all ages. Less 6-OHDA was required in tg(Prnp-SNCA A30P) Tg (1 µg) than in WT (3µg) mice for an ipsilateral rotational bias by d-amphetamine. That was not seen with the Snca(tm(A30P)) strain. A small dose of 6-OHDA (0.33 µg) led to contralateral rotations and elevated striatal DA in Tg/Tm mice of both lines but otherwise 6-OHDA-induced striatal DA depletion was similar in all mice, indicating no A30P-aSyn-related toxin sensitivity. 3,4-Dihydroxyphenylacetic acid/DA-ratio was elevated in tg(Prnp-SNCA A30P) mice, suggesting an enhanced DA turnover. This ratio and homovanillic acid/DA-ratio were declined in Snca(tm(A30P)) mice. Our results demonstrate that the two differently constructed A30P-aSyn mouse strains have distinct behavioral and biochemical characteristics, some of which are opposite. Since the two lines with the same background were not identically produced, the deviations found may be partially caused by factors other than aSyn-related genetic differences.

A mathematical model of adult subventricular neurogenesis.

Neurogenesis has been the subject of active research in recent years and many authors have explored the phenomenology of the process, its regulation and its purported purpose. Recent developments in bioluminescent imaging (BLI) allow direct in vivo imaging of neurogenesis, and in order to interpret the experimental results, mathematical models are necessary. This study proposes such a mathematical model that describes adult mammalian neurogenesis occurring in the subventricular zone and the subsequent migration of cells through the rostral migratory stream to the olfactory bulb (OB). This model assumes that a single chemoattractant is responsible for cell migration, secreted both by the OB and in an endocrine fashion by the cells involved in neurogenesis. The solutions to the system of partial differential equations are compared with the physiological rodent process, as previously documented in the literature and quantified through the use of BLI, and a parameter space is described, the corresponding solution to which matches that of the rodent model. A sensitivity analysis shows that this parameter space is stable to perturbation and furthermore that the system as a whole is sloppy. A large number of parameter sets are stochastically generated, and it is found that parameter spaces corresponding to physiologically plausible solutions generally obey constraints similar to the conditions reported in vivo. This further corroborates the model and its underlying assumptions based on the current understanding of the investigated phenomenon. Concomitantly, this leaves room for further quantitative predictions pertinent to the design of future proposed experiments.

A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease.

BACKGROUND AND PURPOSE: The aggregation of α-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH: Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg(-1) a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS: In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS: The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes.

On the road to leucine-rich repeat kinase 2 signalling: evidence from cellular and in vivo studies.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Although PD has long been considered a purely sporadic disorder, genetic research has revealed an underlying genetic cause in at least 10% of all PD cases. To date, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial PD. Moreover, given the strong clinical and neuropathological similarities between LRRK2 PD and the sporadic forms of the disease, the notion is supported that the unravelling of the molecular pathways underlying LRRK2 PD will greatly contribute to our general understanding of PD. Therefore, intense research efforts have been focused on the understanding of the physiological function of LRRK2 and its relation to PD. To date, progress has been made in these fields based on the study of LRRK2 cell culture models, the identification of LRRK2 interaction partners and kinase substrates and the generation of LRRK2 animal models. In this review, the current insights into the cellular role of LRRK2 are discussed. The overview reveals a potential involvement of LRRK2 in major cell signalling pathways including apoptosis, cytoskeleton dynamics, protein translation, mitogen-activated protein kinase signalling and specific dopaminergic functions, consistent with its proposed role as a signal transduction protein.

Efficient and stable transduction of dopaminergic neurons in rat substantia nigra by rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9.

Dysfunction of the nigrostriatal system is the major cause of Parkinson's disease (PD). This brain region is therefore an important target for gene delivery aiming at disease modeling and gene therapy. Recombinant adeno-associated viral (rAAV) vectors have been developed as efficient vehicles for gene transfer into the central nervous system. Recently, several serotypes have been described, with varying tropism for brain transduction. In light of the further development of a viral vector-mediated rat model for PD, we performed a comprehensive comparison of the transduction and tropism for dopaminergic neurons (DNs) in the adult Wistar rat substantia nigra (SN) of seven rAAV vector serotypes (rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9). All vectors were normalized by titer and volume, and stereotactically injected into the SN. Gene expression was assessed non-invasively and quantitatively in vivo by bioluminescence imaging at 2 and 5 weeks after injection, and was found to be stable over time. Immunohistochemistry at 6 weeks following injection revealed the most widespread enhanced green fluorescence protein expression and the highest number of positive nigral cells using rAAV 2/7, 2/9 and 2/1. The area transduced by rAAV 2/8 was smaller, but nevertheless almost equal numbers of nigral cells were targeted. Detailed confocal analysis revealed that serotype 2/7, 2/9, 2/1 and 2/8 transduced at least 70% of the DNs. In conclusion, these results show that various rAAV serotypes efficiently transduce nigral DNs, but significant differences in transgene expression pattern and level were observed.

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors.

The development of in vivo imaging protocols to reliably track transplanted cells or to report on gene expression is critical for treatment monitoring in (pre)clinical cell and gene therapy protocols. Therefore, we evaluated the potential of lentiviral vectors (LVs) and adeno-associated viral vectors (AAVs) to express the magnetic resonance imaging (MRI) reporter gene ferritin in the rodent brain. First, we compared the induction of background MRI contrast for both vector systems in immune-deficient and immune-competent mice. LV injection resulted in hypointense (that is, dark) changes of T(2)/T(2)(*) (spin-spin relaxation time)-weighted MRI contrast at the injection site, which can be partially explained by an inflammatory response against the vector injection. In contrast to LVs, AAV injection resulted in reduced background contrast. Moreover, AAV-mediated ferritin overexpression resulted in significantly enhanced contrast to background on T(2)(*)-weighted MRI. Although sensitivity associated with the ferritin reporter remains modest, AAVs seem to be the most promising vector system for in vivo MRI reporter gene imaging.

Magnetic resonance imaging and spectroscopy methods for molecular imaging.

Magnetic resonance imaging (MRI), one of the most powerful imaging modalities available for clinical diagnosis, has contributed significantly to phenotyping of transgenic organisms and to cellular imaging and is now gaining importance in the field of molecular imaging. Its advantage is the ability to provide in vivo information with high resolution and good soft tissue contrast as compared to established other molecular imaging methods. MRI can non-invasively report on cell localisation and migration with detailed anatomical background information, which is of great interest in cellular therapies. Recent technological advances and contrast generation strategies aim to bring MRI beyond cellular imaging to the detection of functional changes in vivo. MR based monitoring of molecular processes, requires the development of contrast agents and targeting methods as well as improvements in the methods sensitivity. Here, an overview is provided on advanced MR technologies and contrast generation strategies for this purpose. This includes MRI and MR spectroscopic methods for molecular imaging and various approaches for targeted and responsive contrast generation to visualize functional changes of particular cells. A description of different methods is provided, as well as the potentials and challenges of MR techniques for the visualization of molecular processes in vivo.

In vivo bioluminescence imaging in an experimental mouse model for dendritic cell based immunotherapy against malignant glioma.

The value of bioluminescence imaging (BLI) for experimental cancer models has become firmly established. We applied BLI to the GL261 glioma model in the context of dendritic cell (DC) immunotherapy. Initial validation revealed robust linear correlations between in vivo, ex vivo and in vitro luciferase activity measurements. Ex vivo BLI demonstrated midline crossing and leakage of tumor cells. Orthotopically challenged mice followed with BLI showed an initial adaptation phase, after which imaging data correlated linearly with stereologically determined tumor dimensions. Transition from healthy to moribund state corresponded with an increasing in vivo flux but the onset of neurological deficit was clearly delayed compared to the onset of in vivo flux increase. BLI was implemented in prophylactic immunotherapy and imaging data were prognostic for therapy outcome. Three distinct response patterns were detected. Our data underscore the feasibility of in vivo BLI in an experimental immunotherapeutic setting in the GL261 glioma model.

Alpha-synuclein A30P point-mutation generates age-dependent nigrostriatal deficiency in mice.

Lewy bodies are mainly composed of alpha-synuclein (SNCA) and specific mutations in SNCA gene are related to familial forms of Parkinson's disease (PD). The purpose of our study was to generate a mouse line with A30P knock-in point mutation in SNCA gene and to test if a single point-mutation is able to turn otherwise normal SNCA into a toxic form. The behavioral profile of SNCA A30P mice was followed for 16 months. Generally, these mice are healthy and viable without any obvious abnormalities. Starting from the age of 13 months mice developed a significant deficit in motor performance tests related to nigrostriatal function (ink-test and beam walk). In other tests (motility boxes, rotarod) mice continuously performed normally. Moreover, SNCA A30P mice expressed the altered sensitivity to VMAT2 inhibitor reserpine, possibly reflecting a functional deficiency of dopamine. Indeed, mice at 15 months of age had significantly reduced levels of dopamine and its major metabolite DOPAC in the striatum, and reduced levels of dopamine in the mesolimbic system. The present study confirms that SNCA plays an important role in the development of PD and an insertion of a single point mutation is sufficient to generate age-related decline in specific motor performance. The generated mouse line has a potential to become a model for PD with comparable time course and phenotype.

Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain.

HIV-derived lentiviral vectors are efficient vehicula to deliver genes into the brain and hold great promise for future gene therapy of neurodegenerative disorders. However, administration of the current vector preparations in mouse brain was found to induce a systemic immune response to vector proteins and a modest inflammation in the brain. Moreover, serum antibodies from vector-treated animals were capable of partially neutralizing lentiviral vector-mediated transduction in cell culture. To avoid this unexpected immune reaction, we have optimized new vector production and purification protocols. Purification by sucrose gradient ultracentrifugation abolished the immune response, but vector titers also decreased substantially. Lentiviral vector production in the absence of serum in the cell culture medium equally reduced immunogenicity without affecting transduction efficiency. These results have important implications for future clinical use of lentiviral vectors, and for the use of lentiviral vectors to create animal models for neurodegenerative diseases that have an important neuroinflammatory component.

Neuroprotective gene therapy for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease characterised by a progressive loss of the dopaminergic neurones in the substantia nigra pars compacta. Accumulating evidence indicates that apoptosis contributes to neuronal cell death in PD patients' brain. Excitotoxicity, oxidative stress, and mitochondrial respiratory failure are thought to be the key inducers of the apoptotic cascade. Even though the initial cause and the mechanism of degeneration are poorly understood, neuroprotection can be achieved by interfering with neuronal cell death either directly or by preventing neuronal dysfunction. Potential agents for neuroprotection are neurotrophic factors, inhibitors of apoptosis or anti-oxidative agents. However, the existence of the blood-brain barrier precludes systemic delivery of these factors. In situ gene delivery provides strategies for local and sustained administration of protective factors at physiologically relevant doses. Viral vectors mediating stable gene expression in the central nervous system exist and are still under development. Efficacy of these vectors has repeatedly been demonstrated in the animal models both ex vivo and in vivo. Ex vivo gene delivery could furthermore be combined with cell replacement therapies by transplanting genetically modified cells compensating for the lost neuronal cell population in order to provide neuroprotection to both the grafted cells and degenerating host neurones. However, several aspects of gene transfer, such as uncontrolled diffusion, axonal transport, unpredictable site of integration and immunological responses, still raise safety concerns and justify further development of viral and non-viral vectors as well as genetic elements with tightly controlled gene expression. Various relevant animal models for Parkinson's disease are available for the evaluation of gene therapy strategies. These include induction of cell death in specific neurone population through administration of toxins either directly in the brain or systemically, as well as transgenic mice expressing human disease-associated mutations.

Mice with a homozygous gene trap vector insertion in mgcRacGAP die during pre-implantation development.

In a phenotypic screen in mice using a gene trap approach in embryonic stem cells, we have identified a recessive loss-of-function mutation in the mgcRacGAP gene. Maternal protein is present in the oocyte, and mgcRacGAP gene transcription starts at the four-cell stage and persists throughout mouse pre-implantation development. Total mgcRacGAP deficiency results in pre-implantation lethality. Such E3.5 embryos display a dramatic reduction in cell number, but undergo compaction and form a blastocoel. At E3.0-3.5, binucleated blastomeres in which the nuclei are partially interconnected are frequently observed, suggesting that mgcRacGAP is required for normal mitosis and cytokinesis in the pre-implantation embryo. All homozygous mutant blastocysts fail to grow out on fibronectin-coated substrates, but a fraction of them can still induce decidual swelling in vivo. The mgcRacGAP mRNA expression pattern in post-implantation embryos and adult mouse brain suggests a role in neuronal cells. Our results indicate that mgcRacGAP is essential for the earliest stages of mouse embryogenesis, and add evidence that CYK-4-like proteins also play a role in microtubule-dependent steps in the cytokinesis of vertebrate cells. In addition, the severe phenotype of null embryos indicates that mgcRacGAP is functionally non-redundant and cannot be substituted by other GAPs during early cleavage of the mammalian embryo.