Controlled Striatal DOPA Production From a Gene Delivery System in a Rodent Model of Parkinson's Disease.

Conventional symptomatic treatment for Parkinson's disease (PD) with long-term L-3,4-dihydroxyphenylalanine (DOPA) is complicated with development of drug-induced side effects. In vivo viral vector-mediated gene expression encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) provides a drug delivery strategy of DOPA with distinct advantages over pharmacotherapy. Since the brain alterations made with current gene transfer techniques are irreversible, the therapeutic approaches taken to the clinic should preferably be controllable to match the needs of each individual during the course of their disease. We used a recently described tunable gene expression system based on the use of destabilized dihydrofolate reductase (DD) and generated a N-terminally coupled GCH1 enzyme (DD-GCH1) while the TH enzyme was constitutively expressed, packaged in adeno-associated viral (AAV) vectors. Expression of DD-GCH1 was regulated by the activating ligand trimethoprim (TMP) that crosses the blood-brain barrier. We show that the resulting intervention provides a TMP-dose-dependent regulation of DOPA synthesis that is closely linked to the magnitude of functional effects. Our data constitutes the first proof of principle for controlled reconstitution of dopamine capacity in the brain and suggests that such next-generation gene therapy strategies are now mature for preclinical development toward use in patients with PD.

Contrasting walking styles map to discrete neural substrates in the mouse brainstem.

Walking is a slow gait which is particularly adaptable to meet internal or external needs and is prone to maladaptive alterations that lead to gait disorders. Alterations can affect speed, but also style (the way one walks). While slowed speed may signify the presence of a problem, style represents the hallmark essential for clinical classification of gait disorders. However, it has been challenging to objectively capture key stylistic features while uncovering neural substrates driving these features. Here we revealed brainstem hotspots that drive strikingly different walking styles by employing an unbiased mapping assay that combines quantitative walking signatures with focal, cell type specific activation. We found that activation of inhibitory neurons that mapped to the ventromedial caudal pons induced slow motion-like style. Activation of excitatory neurons that mapped to the ventromedial upper medulla induced shuffle-like style. Contrasting shifts in walking signatures distinguished these styles. Activation of inhibitory and excitatory neurons outside these territories or of serotonergic neurons modulated walking speed, but without walking signature shifts. Consistent with their contrasting modulatory actions, hotspots for slow-motion and shuffle-like gaits preferentially innervated different substrates. These findings lay the basis for new avenues to study mechanisms underlying (mal)adaptive walking styles and gait disorders.

Shifts in Gait Signatures Mark the End of Lifespan in Mice, With Sex Differences in Timing.

Reduced walking speed is a hallmark of functional decline in aging across species. An age-related change in walking style may represent an additional key marker signifying deterioration of the nervous system. Due to the speed dependence of gait metrics combined with slowing of gait during aging, it has been challenging to determine whether changes in gait metrics represent a change in style. In this longitudinal study we employed gait signatures to separate changes in walking style and speed in mice. We compared gait signatures at mature adult age with middle aged, old and geriatric time points and included female and male sub-cohorts to examine sex differences in nature or timing signature shifts. To determine whether gait signature shifts occurred independently from a decline in other mobility domains we measured balance and locomotor activity. We found that walking speed declined early, whereas gait signatures shifted very late during the aging process. Shifts represented longer swing time and stride length than expected for speed, as in slow motion, and were preceded by a decline in other mobility domains. The pattern of shifts was similar between female and male cohorts, but with sex differences in timing. We conclude that changes in walking style, speed and other mobility domains represent separate age-related phenomena. These findings call for careful, sex specific selection of type and timing of outcome measures in mechanistic or interventional studies. The pattern of age-related gait signature shifts is distinct from patterns seen in neurodegenerative conditions and may be a translatable marker for the end of the lifespan.

The RGD-containing peptide fragment of osteopontin protects tyrosine hydroxylase positive cells against toxic insult in primary ventral mesencephalic cultures and in the rat substantia nigra.

We have previously shown that the multi-functional phosphoprotein osteopontin (OPN) is present in the substantia nigra (SN) and that its mRNA and protein expression are up-regulated following toxic insult. We now report the effects of the arginine-glycine-aspartic acid (RGD)-containing peptide fragment of OPN and OPN inactivation on the survival of tyrosine hydroxylase (TH) positive neurones in primary rat ventral mesencephalic (VM) cultures and in SN in the rat. Treatment of VM cultures with the fragment of OPN containing the RGD integrin binding domain did not decrease TH positive cell number, but instead the peptide fragment protected against cell loss induced by both MPP(+) and lipopolysaccharide (LPS). Incorporation of an OPN antibody into VM cultures caused a concentration-dependent loss of TH positive neurones. The OPN antibody also exacerbated MPP(+) - and LPS-induced cell loss at all concentrations tested. In the rat, administration of the RGD-containing peptide fragment of OPN protected TH positive neurones against a mechanically-induced lesion and against 6-hydroxydopamine- and LPS-induced cell loss. The protection against 6-hydroxydopamine toxicity was confirmed in a separate study using stereological analysis. By contrast, stereotaxic injection of the OPN antibody into the SN resulted in a loss of TH positive cells. These results suggest that OPN may be necessary for the survival of TH positive cells in SN but through the RGD-containing peptide fragment may also have neuroprotective properties relevant to Parkinson's disease.

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease.

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b - anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.

Translational methods to detect asymmetries in temporal and spatial walking metrics in parkinsonian mouse models and human subjects with Parkinson's disease.

Clinical signs in Parkinson's disease (PD), including parkinsonian gait, are often asymmetric, but mechanisms underlying gait asymmetries in PD remain poorly understood. A translational toolkit, a set of standardized measures to capture gait asymmetries in relevant mouse models and patients, would greatly facilitate research efforts. We validated approaches to quantify asymmetries in placement and timing of limbs in mouse models of parkinsonism and human PD subjects at speeds that are relevant for human walking. In mice, we applied regression analysis to compare left and right gait metrics within a condition. To compare alternation ratios of left and right limbs before and after induction of parkinsonism, we used circular statistics. Both approaches revealed asymmetries in hind- and forelimb step length in a unilateral PD model, but not in bilateral or control models. In human subjects, a similar regression approach showed a step length asymmetry in the PD but not control group. Sub-analysis of cohorts with predominant postural instability-gait impairment and with predominant tremor revealed asymmetries for step length in both cohorts and for swing time only in the former cohort. This translational approach captures asymmetries of gait in mice and patients. Application revealed striking differences between models, and that spatial and temporal asymmetries may occur independently. This approach will be useful to investigate circuit mechanisms underlying the heterogeneity between models.

A translational approach to capture gait signatures of neurological disorders in mice and humans.

A method for capturing gait signatures in neurological conditions that allows comparison of human gait with animal models would be of great value in translational research. However, the velocity dependence of gait parameters and differences between quadruped and biped gait have made this comparison challenging. Here we present an approach that accounts for changes in velocity during walking and allows for translation across species. In mice, we represented spatial and temporal gait parameters as a function of velocity and established regression models that reproducibly capture the signatures of these relationships during walking. In experimental parkinsonism models, regression curves representing these relationships shifted from baseline, implicating changes in gait signatures, but with marked differences between models. Gait parameters in healthy human subjects followed similar strict velocity dependent relationships which were altered in Parkinson's patients in ways that resemble some but not all mouse models. This novel approach is suitable to quantify qualitative walking abnormalities related to CNS circuit dysfunction across species, identify appropriate animal models, and it provides important translational opportunities.

Increased neurotrophic factor levels in ventral mesencephalic cultures do not explain the protective effect of osteopontin and the synthetic 15-mer RGD domain against MPP+ toxicity.

The synthetic 15-mer arginine-glycine-aspartic acid (RGD) domain of osteopontin (OPN) is protective in vitro and in vivo against dopaminergic cell death and this protective effect may be mediated through interaction with integrin receptors to regulate neurotrophic factor levels. We now examine this concept in rat primary ventral mesencephalic (VM) cultures. 1-Methyl-4-phenylpyridinium (MPP+) exposure reduced tyrosine hydroxylase (TH)-positive cell number and activated glial cells as shown by increased glial fibrillary acidic protein (GFAP), oxycocin-42 (OX-42) and ectodermal dysplasia 1 (ED-1) immunoreactivity. Both OPN and the RGD domain of OPN were equally protective against MPP+ toxicity in VM cultures and both increased glial-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) levels. The effects of OPN and the RGD domain were accompanied by a decrease in numbers of activated microglia but with no change in astrocyte number. However, full-length OPN and the RGD domain of OPN remained protective against MPP+ toxicity in the presence of a GDNF neutralising antibody. This suggests that increased GDNF levels do not underlie the protective effect observed with OPN. Rather, OPN's protective effect may be mediated through decreased glial cell activation.

Neuroprotection by the selective iNOS inhibitor GW274150 in a model of Parkinson disease.

Neuroinflammation and the activation of inducible nitric oxide synthase (iNOS) have been proposed to play a role in the pathogenesis of Parkinson disease (PD). In this study we investigated the effects of the selective iNOS inhibitor GW274150 in the 6-OHDA model of PD. 6-OHDA administration was associated with increased numbers of cells expressing iNOS. Administration of the iNOS inhibitor twice daily for 7 days, beginning 2 days after the 6-OHDA lesioning, led to a significant neuroprotection as shown by assessment of the integrity of the nigrostriatal system by tyrosine hydroxylase immunocytochemistry and HPLC assessment of striatal dopamine content. However, GW274150 displayed a bell-shaped neuroprotective profile, being ineffective at high doses. 6-OHDA lesioning was associated with an increase in microglial activation as assessed by the MHC II antigen OX-6 and the number of matrix metalloproteinase 9 (MMP-9)-immunopositive cells. NO is a known modulator of MMP-9, and iNOS inhibition was associated with decreased numbers of MMP-9-immunopositive cells, culminating in a reduction in the numbers of reactive microglia. Withdrawal of GW274150 for a further 7 days negated any neuroprotective effects of iNOS inhibition, suggesting that the damaging effects of inflammation last beyond 7 days in this model and the continued administration of the drug may be required.