The contribution of inflammatory astrocytes to BBB impairments in a brain-chip model of Parkinson's disease.

Astrocyte dysfunction has previously been linked to multiple neurodegenerative disorders including Parkinson's disease (PD). Among their many roles, astrocytes are mediators of the brain immune response, and astrocyte reactivity is a pathological feature of PD. They are also involved in the formation and maintenance of the blood-brain barrier (BBB), but barrier integrity is compromised in people with PD. This study focuses on an unexplored area of PD pathogenesis by characterizing the interplay between astrocytes, inflammation and BBB integrity, and by combining patient-derived induced pluripotent stem cells with microfluidic technologies to generate a 3D human BBB chip. Here we report that astrocytes derived from female donors harboring the PD-related LRRK2 G2019S mutation are pro-inflammatory and fail to support the formation of a functional capillary in vitro. We show that inhibition of MEK1/2 signaling attenuates the inflammatory profile of mutant astrocytes and rescues BBB formation, providing insights into mechanisms regulating barrier integrity in PD. Lastly, we confirm that vascular changes are also observed in the human postmortem substantia nigra of both males and females with PD.

Cysteamine as a novel disease-modifying compound for Parkinson's disease: Over a decade of research supporting a clinical trial.

To date, medical and surgical interventions offered to patients with Parkinson's disease (PD) serve only to manage clinical symptoms; they have not shown the capacity to halt nor reverse degenerative processes. There is therefore an urgent need to identify and/or develop therapeutic strategies that will demonstrate 'disease modifying' capacities. The molecule cystamine, and its reduced form cysteamine, act via a number of pathways determined to be critical to the pathogenesis of PD. In particular, cystamine is capable of crossing the blood-brain barrier, and both agents (cystamine and cysteamine) can promote the secretion of neurotrophic factors, inhibit oxidative stress, reduce inflammatory responses and importantly, have already been trialed in humans for a number of other clinical indications. In the last decade, our laboratory has accumulated compelling evidence that both cystamine and cysteamine can halt, and even reverse, ongoing neurodegenerative processes in a number of different models of PD, and as such, should now be taken forward to clinical trials in PD.

A brief history of antibody-based therapy.

Active and passive immunization have been used to treat human disease for hundreds of years and improvements in technology and knowledge is only increasing the number of therapeutic applications. The current and future use of immunization to treat neurodegenerative diseases are briefly described herein to serve as an introduction to this special issue.

GSK-3ß-induced Tau pathology drives hippocampal neuronal cell death in Huntington's disease: involvement of astrocyte-neuron interactions.

Glycogen synthase kinase-3ß (GSK-3ß) has emerged as a critical factor in several pathways involved in hippocampal neuronal maintenance and function. In Huntington's disease (HD), there are early hippocampal deficits both in patients and transgenic mouse models, which prompted us to investigate whether disease-specific changes in GSK-3ß expression may underlie these abnormalities. Thirty-three postmortem hippocampal samples from HD patients (neuropathological grades 2-4) and age- and sex-matched normal control cases were analyzed using real-time quantitative reverse transcription PCRs (qPCRs) and immunohistochemistry. In vitro and in vivo studies looking at hippocampal pathology and GSK-3ß were also undertaken in transgenic R6/2 and wild-type mice. We identified a disease and stage-dependent upregulation of GSK-3ß mRNA and protein levels in the HD hippocampus, with the active isoform pGSK-3ß-Tyr(216) being strongly expressed in dentate gyrus (DG) neurons and astrocytes at a time when phosphorylation of Tau at the AT8 epitope was also present in these same neurons. This upregulation of pGSK-3ß-Tyr(216) was also found in the R6/2 hippocampus in vivo and linked to the increased vulnerability of primary hippocampal neurons in vitro. In addition, the increased expression of GSK-3ß in the astrocytes of R6/2 mice appeared to be the main driver of Tau phosphorylation and caspase3 activation-induced neuronal death, at least in part via an exacerbated production of major proinflammatory mediators. This stage-dependent overactivation of GSK-3ß in HD-affected hippocampal neurons and astrocytes therefore points to GSK-3ß as being a critical factor in the pathological development of this condition. As such, therapeutic targeting of this pathway may help ameliorate neuronal dysfunction in HD.

Cystamine/cysteamine rescues the dopaminergic system and shows neurorestorative properties in an animal model of Parkinson's disease.

The neuroprotective properties of cystamine identified in pre-clinical studies have fast-tracked this compound to clinical trials in Huntington's disease, showing tolerability and benefits on motor symptoms. We tested whether cystamine could have such properties in a Parkinson's disease murine model and now provide evidence that it can not only prevent the neurodegenerative process but also can reverse motor impairments created by a 6-hydroxydopamine lesion 3 weeks post-surgery. Importantly, we report that cystamine has neurorestorative properties 5 weeks post-lesion as seen on the number of nigral dopaminergic neurons which is comparable with treatments of cysteamine, the reduced form of cystamine used in the clinic, as well as rasagiline, increasingly prescribed in early parkinsonism. All three compounds induced neurite arborization of the remaining dopaminergic cells which was further confirmed in ex vivo dopaminergic explants derived from Pitx3-GFP mice. The disease-modifying effects displayed by cystamine/cysteamine would encourage clinical testing.

Single-cell suspension methodology favors survival and vascularization of fetal striatal grafts in the YAC128 mouse model of Huntington's disease.

Cell replacement therapies have yielded variable and short-lived benefits in Huntington's disease (HD) patients. This suboptimal outcome is likely due to the fact that graft survival is compromised long term because grafts are subjected to a host's microglial inflammatory response, to a lack of adequate trophic support, and possibly to cortical excitotoxicity. However, graft demise may also relate to more straightforward issues such as cell preparation methodology (solid grafts vs. cell suspension). Indeed, we recently reported that solid grafts are poorly revascularized in HD patients transplanted 9 and 12 years previously. To evaluate whether methodological issues relating to cell preparation may have an impact on graft viability, we implanted green fluorescent protein (GFP(+)) single-cell suspensions of fetal striatal neuronal cells into the striatum of YAC128 HD mice. Postmortem evaluation yielded comparable graft survival in YAC128 mice and their wild-type littermates (noncarrier) at 1 and 3 months posttransplantation. Additionally, the degrees of graft revascularization in the YAC128 and noncarrier mice were similar, with both capillaries and large-caliber vessels observable within the grafted tissue. Furthermore, GFP(+) cells interacted well with host blood vessels, indicating integration of the donor cells within the recipient brain. These observations, combined with our recent report of poor revascularization of solid grafts in the HD-transplanted patients, suggest that the success of cell transplantation can be improved by optimizing methodological aspects relating to cell preparation.

The fate of cell grafts for the treatment of Huntington's disease: the post-mortem evidence.

The hope that cell transplantation therapies will provide an ideal treatment option for neurodegenerative diseases has been considerably revived with the remarkable advancements in genetic engineering towards active cell fate determination in vitro. However, for disorders such as Huntington's disease (HD), the challenges that we face are still enormous. This autosomal dominant genetic disorder leads, in part, to massive neuronal loss and severe brain atrophy which, despite the cell type used, cannot be easily repaired. And before large clinical trials are even considered, we must take a critical look at the outcomes of the pilot studies already available, not only from a clinical perspective but also by a careful assessment of what we can learn from the autopsies of HD patients who have undergone transplantation. In this review, we summarize and discuss the seven transplantation pilot trials that were initiated worldwide in HD patients more than a decade ago, with a particular emphasis on the post-mortem analyses of nine unique cases. Moreover, we describe a series of factors, both technical and related to patient selection, that we deem important to predict the outcome of cell grafts in HD therapy.

An in vitro perspective on the molecular mechanisms underlying mutant huntingtin protein toxicity.

Huntington's disease (HD) is a devastating neurodegenerative disorder whose main hallmark is brain atrophy. However, several peripheral organs are considerably affected and their symptoms may, in fact, manifest before those resulting from brain pathology. HD is of genetic origin and caused by a mutation in the huntingtin gene. The mutated protein has detrimental effects on cell survival, but whether the mutation leads to a gain of toxic function or a loss of function of the altered protein is still highly controversial. Most currently used in vitro models have been designed, to a large extent, to investigate the effects of the aggregation process in neuronal-like cells. However, as the pathology involves several other organs, new in vitro models are critically needed to take into account the deleterious effects of mutant huntingtin in peripheral tissues, and thus to identify new targets that could lead to more effective clinical interventions in the early course of the disease. This review aims to present current in vitro models of HD pathology and to discuss the knowledge that has been gained from these studies as well as the new in vitro tools that have been developed, which should reflect the more global view that we now have of the disease.

MyD88 deficiency results in both cognitive and motor impairments in mice.

The myeloid differentiation primary response gene 88 (MyD88) product is the most common adaptor protein implicated in Toll-like and interleukin receptor (TIR) domain signaling and thus plays an important role in the innate immune system. Despite the fact that the MyD88-dependent pathway has emerged as an important player in cell death processes described in several animal models of neurodegenerative disorders, the contribution of this pathway to specific behavioral phenotypes has been largely ignored. To understand the full implication of this pathway, we tested MyD88(-/-) mice for both motor and cognitive functions in normal conditions. MyD88(-/-) mice displayed impaired spatial and working memory as detected by the Barnes maze, the water T-maze and the passive avoidance tests. Furthermore, MyD88(-/-) mice demonstrated hypolocomotion in the open-field and wheel activity systems, as well as impairments in motor coordination and balance using the pole test and the rotarod. Our findings shed light on behavioral alterations that are associated with the deletion of the MyD88 protein in physiological conditions. These behavioral effects should be taken into consideration when assessing the role of the MyD88-dependent pathway in various infectious and non-infectious conditions.

High-fat diet exacerbates MPTP-induced dopaminergic degeneration in mice.

The identification of modifiable nutritional risk factors is highly relevant to the development of preventive strategies for neurodegenerative disorders including Parkinson's disease (PD). In this study, adult C57BL/6 mice were fed either a control (CD-12%kcal) or a high-fat diet (HFD-60%kcal) for 8 weeks prior to MPTP exposure, a toxin which recreates a number of pathological features of PD. HFD-fed mice significantly gained weight (+41%), developed insulin resistance and a systemic immune response characterized by an increase in circulating leukocytes and plasmatic cytokines/chemokines (interleukin-1α, MCP-1, MIP-1α). As expected, the MPTP treatment produced nigral dopaminergic degeneration as evidenced by the loss of striatal dopamine and the decreased number of nigral tyrosine hydroxylase (TH)- and dopamine transporter-expressing neurons (23% and 25%, respectively). However, exposure to HFD exacerbated the effects of MPTP on striatal TH (23%) and dopamine levels (32%), indicating that diet-induced obesity is associated with a reduced capacity of nigral dopaminergic terminals to cope with MPTP-induced neurotoxicity. Since high-fat consumption is commonplace in our modern society, dietary fat intake may represent an important modifiable risk factor for PD.

The role of immunity in Huntington's disease.

Huntington's disease (HD) is a devastating and incurable neurodegenerative disorder characterized by progressive cognitive, psychiatric and motor impairments. Although the disease has been seen as a disorder purely of the brain, there is now emerging evidence that abnormalities outside the central nervous system are commonly seen in HD. Indeed, the mutant huntingtin (mHtt) coded for by the abnormal gene in HD is found in every cell type where its presence has been sought. In particular, there are a number of recent observations in HD patients that mHtt interacts with the immune system with accumulating evidence that changes in the immune system may critically contribute to the pathology of HD. However, the nature of this contribution remains unclear, to the extent that it is not even known whether the immune system has a beneficial or detrimental role in HD patients. In this review, we attempt to bring a novel understanding to the interaction of the immune system to HD pathology, thereby shedding light on its potential pathogenic role. As part of this discussion, we revisit the clinical data on the anti-inflammatory drug trials in HD and propose new experimental approaches to interrogate the role of immunity in this currently incurable disorder.

The critical role of the MyD88-dependent pathway in non-CNS MPTP-mediated toxicity.

A growing body of evidence supports a role of inflammation in the loss of central nervous system neurons both to acute and chronic insults, while its contribution to the loss of neurons in the enteric nervous system remains largely uninvestigated. We have addressed this issue by exploring the role of inflammation in dopaminergic (DAergic) myenteric neuronal degeneration secondary to MPTP lesioning in mice deficient in MyD88, a protein implicated in the cascade of events leading to the innate immune response. Our results show that MPTP-treated MyD88 knock out (MyD88(-/-)) mice were protected against the toxin-induced TH-immunoreactive neuronal degeneration at the level of the myenteric plexus of the distal ileum, which causes a 50% loss of such neurons in MPTP-treated WT mice. Interestingly, the density of macrophages was the same in the MyD88(-/-) mice subjected to MPTP, as opposed to the increase in density observed in wild-type (WT) mice treated with the toxin, which was due to an infiltration of monocyte from the blood to the myenteric tissue. Furthermore, in MPTP-treated MyD88(-/-) mice, resident macrophages exhibited a predominant pro-repair phenotype, which could have contributed to the protection of DAergic neurons in the myenteric plexus. Taken together, our results suggest a critical role for the MyD88-dependent pathway in the gastrointestinal DAergic degeneration induced by MPTP.

Potential of cystamine and cysteamine in the treatment of neurodegenerative diseases.

Neurodegenerative disorders are a subset of disabling pathologies characterized, in part, by a progressive and specific loss of certain brain cell populations. Current therapeutic approaches for the treatment of these disorders are mainly designed towards symptom management and do not manifestly block their typified neuronal loss. However, research conducted over the past decade has reflected the increasing interest and need to find disease-modifying molecules. Among the several neuroprotective agents emerging from experimental animal work, cystamine, as well as its reduced form cysteamine, have been identified as potential candidate drugs. Given the significant benefits observed in a Huntington's disease (HD) model, cysteamine has recently leaped to clinical trial. Here, we review the beneficial properties of these compounds as reported in animal studies, their mechanistic underpinnings, and their potential implications for the future treatment of patients suffering from neurodegenerative diseases, and more specifically for HD and Parkinson's disease (PD).

Cystamine prevents MPTP-induced toxicity in young adult mice via the up-regulation of the brain-derived neurotrophic factor.

Preclinical data suggest that cystamine stands as a promising neuroprotective agent against Huntington's and Parkinson's diseases. To decipher the mechanisms of action of cystamine, we investigated the effects of various doses of cystamine (10, 50, and 200mg/kg) on the regulation of the brain-derived neurotrophic factor (BDNF), its receptor tropomyosin-receptor-kinase B (TrkB) and on the heat shock protein 70 (Hsp70) brain mRNA expression in relation to the time after administration. We have determined that the lower cystamine dose is the most efficient to promote putative neuroprotective effects. Indeed, an acute administration of 10mg/kg of cystamine increased the expression of BDNF mRNA in the substantia nigra compacta (SNc), although it did not significantly influence TrkB or Hsp70 mRNA. Higher cystamine doses resulted in the absence of activation of any of these markers or led to non-specific effects. We have also substantiated the neuroprotective effect of a 21-day treatment of 10mg/kg/day of cystamine in young adult mice against MPTP-induced loss of tyrosine hydroxylase-striatal fiber density, nigral dopamine cells and nigral Nurr1 mRNA expression. The neuroprotective action of cystamine in the same animals was associated with an up-regulation of BDNF in the SNc. Taken together, these results strengthen the neuroprotective potential of cystamine in the treatment of Parkinson's disease and point towards the up-regulation of BDNF as an important mechanism of action.

Neural transplants in patients with Huntington's disease undergo disease-like neuronal degeneration.

The clinical evaluation of neural transplantation as a potential treatment for Huntington's disease (HD) was initiated in an attempt to replace lost neurons and improve patient outcomes. Two of 3 patients with HD reported here, who underwent neural transplantation containing striatal anlagen in the striatum a decade earlier, have demonstrated marginal and transient clinical benefits. Their brains were evaluated immunohistochemically and with electron microscopy for markers of projection neurons and interneurons, inflammatory cells, abnormal huntingtin protein, and host-derived connectivity. Surviving grafts were identified bilaterally in 2 of the subjects and displayed classic striatal projection neurons and interneurons. Genetic markers of HD were not expressed within the graft. Here we report in patients with HD that (i) graft survival is attenuated long-term; (ii) grafts undergo disease-like neuronal degeneration with a preferential loss of projection neurons in comparison to interneurons; (iii) immunologically unrelated cells degenerate more rapidly than the patient's neurons, particularly the projection neuron subtype; (iv) graft survival is attenuated in the caudate in comparison to the putamen in HD; (v) glutamatergic cortical neurons project to transplanted striatal neurons; and (vi) microglial inflammatory changes in the grafts specifically target the neuronal components of the grafts. These results, when combined, raise uncertainty about this potential therapeutic approach for the treatment of HD. However, these observations provide new opportunities to investigate the underlying mechanisms involved in HD, as well as to explore additional therapeutic paradigms.

Modulation of brain-derived neurotrophic factor as a potential neuroprotective mechanism of action of omega-3 fatty acids in a parkinsonian animal model.

While we recently reported the beneficial effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs) in a mouse model of Parkinson's disease (PD), the mechanisms of action remain largely unknown. Here, we specifically investigated the contribution of the brain-derived neurotrophic factor (BDNF) to the neuroprotective effect of n-3 PUFA observed in a mouse model of PD generated by a subacute exposure to MPTP using a total of 7 doses of 20mg/kg over 5 days. The ten-month high n-3 PUFA treatment which preceded the MPTP exposure induced an increase of BDNF mRNA expression in the striatum, but not in the motor cortex of animals fed the high n-3 PUFA diet. In contrast, n-3 PUFA treatment increased BDNF protein levels in the motor cortex of MPTP-treated mice, an effect not observed in vehicle-treated mice. The mRNA expression of the high-affinity BDNF receptor tropomyosin-related kinase B (TrkB) was increased in the striatum of MPTP-treated mice fed the high n-3 PUFA diet compared to vehicle and MPTP-treated mice on the control diet and to vehicle mice on the high n-3 PUFA diet. These data suggest that the modulation of BDNF expression contributes, in part, to n-3 PUFA-induced neuroprotection in an animal model of PD.

Beneficial effects of dietary omega-3 polyunsaturated fatty acid on toxin-induced neuronal degeneration in an animal model of Parkinson's disease.

In this study, we examined whether omega-3 (n-3) polyunsaturated fatty acids (PUFAs) may exert neuroprotective action in Parkinson's disease, as previously shown in Alzheimer's disease. We exposed mice to either a control or a high n-3 PUFA diet from 2 to 12 months of age and then treated them with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 140 mg/kg in 5 days). High n-3 PUFA dietary consumption completely prevented the MPTP-induced decrease of tyrosine hydroxylase (TH)-labeled nigral cells (P<0.01 vs. MPTP mice on control diet), Nurr1 mRNA (P<0.01 vs. MPTP mice on control diet), and dopamine transporter mRNA levels (P<0.05 vs. MPTP mice on control diet) in the substantia nigra. Although n-3 PUFA dietary treatment had no effect on striatal dopaminergic terminals, the high n-3 PUFA diet protected against the MPTP-induced decrease in dopamine (P<0.05 vs. MPTP mice on control diet) and its metabolite dihydroxyphenylacetic acid (P<0.05 vs. MPTP mice on control diet) in the striatum. Taken together, these data suggest that a high n-3 PUFA dietary intake exerts neuroprotective actions in an animal model of Parkinsonism.

Immune parameters relevant to neural xenograft survival in the primate brain.

The lack of supply and access to human tissue has prompted the development of xenotransplantation as a potential clinical modality for neural cell transplantation. The goal of the present study was to achieve a better understanding of the immune factors involved in neural xenograft rejection in primates. Initially, we quantified complement mediated cell lysis of porcine fetal neurons by primate serum and demonstrated that anti-C5 antibody treatment inhibited cell death. We then developed an immunosuppression protocol that included in vivo anti-C5 monoclonal antibody treatment, triple drug therapy (cyclosporine, methylprednisolone, azathioprine) and donor tissue derived from CD59 or H-transferase transgenic pigs and applied it to pig-to-primate neural cell transplant models. Pre-formed alphaGal, induced alphaGal and primate anti-mouse antibody (PAMA) titers were monitored to assess the immune response. Four primates were transplanted. The three CD59 neural cell recipients showed an induced anti-alphaGal response, whereas the H-transferase neural cell recipient exhibited consistently low anti-alphaGal titers. Two of these recipients contained surviving grafts as detected by immunohistochemistry using selected neural markers. Graft survival correlated with high dose cyclosporine treatment, complete complement blockade and the absence of an induced PAMA response to the murine anti-C5 monoclonal antibodies.

Combined inhibition of apoptosis and complement improves neural graft survival of embryonic rat and porcine mesencephalon in the rat brain.

To define potential mechanisms of cell death during neural cell transplantation, we investigated the role of intracellular caspase activation in combination with the activation of serum complement. We demonstrated that ventral mesencephalic (VM) cells are susceptible to complement-mediated cell lysis that can be blocked with an anti-C5 complement inhibitor (18A10). We also determined that incubating freshly isolated allogenic VM cells with the caspase inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF), followed by immediate striatal implantation, led to a 2.5-fold increase in tyrosine hydroxylase (TH) cell survival 12 weeks postimplantation (P < 0.05). In contrast, overnight incubation with BAF followed by striatal implantation led to a 2-fold reduction in TH cell survival at 12 weeks (P < 0.05). Using the optimal BAF treatment and complement inhibition, we tested the hypothesis that these treatments would lead to increased cell survival in both allogeneic and xenogeneic transplantation models. We transplanted cell suspensions of (a) rat E14 VM or VM treated with (b) BAF alone, (c) anti-C5, or (d) a combination of BAF and anti-C5. There was a significant increase in the relative number of TH-positive cells in the BAF/anti-C5 group versus control at 12 weeks posttransplantation. Similar results were achieved in a pig to rat xenotransplant paradigm. A neuronal xenograft marker (70-kDa neurofilament) also demonstrated relative increases in graft volume in the BAF/anti-C5 treatment group. These studies indicate that more than one mechanism can mediate cell death during neural cell transplantation and that a combined treatment using caspase and complement inhibition can significantly improve cell survival.