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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Immunocytochemical localization of proteoglycans within normal elastin fibers.

Appreciable amounts of glycosaminoglycans have been found by immunocytochemistry within mature elastin fibers of human dermis. On thin sections, elastin fibers showed antigenic sites for monoclonal antibodies recognizing the unsaturated units remaining after digestion of hyaluronic acid with Streptomyces hyaluronidase and after digestion of dermatan and chondroitin sulfates with chondroitinase ABC. Moreover, sectioned elastin fibers were positive towards antibodies raised against synthetic peptides corresponding to amino acid sequences near the N-terminus of the protein core of small matrix proteoglycans PGI and PGII, respectively (Fisher et al., J. Biol. Chem. 262, 9702-9708 (1987)). This is the first demonstration that highly hydrophylic molecules are strictly associated with normally cross-linked elastin. The presence of highly hydrated molecules within the elastin polymer could greatly influence its physiological properties and behavior in pathology.

Differential expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor subunits by calretinin-immunoreactive neurons in the human striatum.

We recently reported the existence of medium and large intemeurons immunoreactive for the calcium-binding protein calretinin in the human striatum. We also showed a selective sparing of all medium, but not all large, calretinin-immunoreactive striatal neurons in Huntington's disease striatum. Because glutamate receptor-mediated excitotoxicity has been implicated in the massive loss of striatal projection neurons that characterizes Huntington's disease, we have applied a double-antigen localization procedure to post mortem tissue from eight normal human subjects to determine the expression of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor subunits 1/2/4 by the calretinin-immunoreactive interneurons. The two types of calretinin-immunoreactive neurons were found to display various patterns of glutamate receptor subunit expression and a specific regionalization was also noted in the expression of these glutamate receptor subunits. Approximately half of the large calretinin-immunoreactive neurons displayed immunoreactivity for glutamate receptor subunits 1 and 2, and about the same proportion of medium calretinin-immunoreactive neurons expressed glutamate receptor subunits 1 and 4. These double-labeled neurons were rather uniformly distributed in the caudate nucleus and putamen. In contrast, as much as 70.1% of the large calretinin-immunoreactive neurons displayed glutamate receptor subunit 4 immunoreactivity in the postcommissural portion of the putamen, an area that corresponds to the sensorimotor striatal territory. For their part, the medium calretinin-immunoreactive neurons were markedly enriched with glutamate receptor subunit 2, 76% of them being double labeled in the caudate nucleus, which corresponds to the striatal associative territory, compared with 85.5% in the postcommissural putamen. Receptor subunit composition plays a key role in determining the functional properties of glutamate receptors, including their permeability to calcium and susceptibility to excitotoxic insults. Thus, the differential expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate glutamate receptor subunits reported here may help to explain the selective sparing of certain types of calretinin-immunoreactive striatal interneurons in Huntington's disease, although other factors, such as post-transcriptional editing, are also likely to be involved.

Enhanced axonal growth from fetal human bcl-2 transgenic mouse dopamine neurons transplanted to the adult rat striatum.

Embryonic neurons transplanted to the adult CNS extend axons only for a developmentally defined period. There are certain intercellular factors that control the axonal extension, one of which may be the expression of the bcl-2 protein. In this study, rats with complete striatal dopamine fiber denervation received embryonic day 14 mouse ventral mesencephalon cells overexpressing human bcl-2 or control wild-type ventral mesencephalon cells. All rats were treated with cyclosporine to prevent rejection and the surviving grafts were analyzed for cell survival and outgrowth of dopaminergic fibers. The results demonstrate that bcl-2 overexpression does not enhance neuronal graft survival. However, the bcl-2 overexpressing neurons had a higher number of dopaminergic fibers that grew longer distances. These results show that overexpression of bcl-2 can result in longer distance axonal growth of transplanted fetal dopaminergic neurons and that genetic modification of embryonic donor cells may enhance their ability to reinnervate a neuronal target territory.

Aging of the human synovium: an in vivo and ex vivo morphological study.

Age-associated changes of the human synovium have been investigated by microarthroscopy, optical and electron microscopy, immunohistochemistry, and cytochemistry. The knee joints of nineteen 15- to 56-year-old subjects, classified as normal by inspection, were carefully examined by microarthroscopy; small synovial tissue biopsy specimens from both the suprapatellar pouch and the medial tibiofemoral gutter were taken. Microarthroscopy showed that the villi were more numerous and the vascular network and cell distribution and profiles less regular in aged individuals. These data were confirmed by scanning electron microscopy, which also showed large areas of the synovial surface devoid of cells and collagen bundles in contact with the joint cavity in aged subjects. Light and transmission electron microscopy confirmed these data and allowed evaluation of the number, distribution, shape, and internal organization of cells as well as the distribution of vessels and the organization of the extracellular matrix in the full thickness of the synovium (down to 2 mm). Particular attention was paid to synovial lining cells, among which three main phenotypes could be recognized: synthetic type (present at all ages and hypertrophied in aged subjects), macrophagelike (increasing with age), and fibroblastlike. Collagen increased with age. Further studies are needed for comprehensive understanding of age-associated changes in the human synovium.

Calretinin gene expression in the human thalamus.

The localization and levels of expression of the calcium-binding protein calretinin (CR) in the human thalamus was studied with an in situ hybridization method applied to formalin-fixed postmortem material from normal individuals. The riboprobe used was generated from a specific fragment of human CR cDNA. As visualized on X-ray film autoradiographs, high levels of CR gene transcript occurred in several thalamic nuclei, including the reticular nucleus, mediodorsal nucleus, rostral intralaminar nuclei (paracentral, central medial and central lateral) and several midline nuclei (paraventricular, reuniens and medioventral nuclei). In the reticular nucleus, neurons expressing CR mRNA were few in number but formed dense and widely distributed clusters. In contrast, virtually all neurons in the rostral intralaminar and midline nuclei expressed very high levels of CR mRNA and formed a prominent rim around the mediodorsal nucleus, which contained scattered clusters of labeled neurons. The caudal intralaminar nuclei, principally the centromedian nucleus, displayed very few neurons expressing CR mRNA. Only the medial part of the parafascicular nucleus expressed moderate levels of CR mRNA. The nuclei of the ventral group (ventral anterior, lateral and posterior nuclei) were virtually devoid of CR gene transcript. This highly heterogeneous pattern of mRNA expression suggests that CR may be heavily involved in the function of the so-called non-specific nuclei, but not in that of the specific relay nuclei of the human thalamus. The data also demonstrate that the presence of CR gene transcript can easily be detected on formalin-fixed sections of the human brain.

Striatal neurones displaying substance P (NK1) receptor immunoreactivity in human and non-human primates.

The striatum of normal human subjects and that of squirrel monkeys (Saimiri sciureus) was found to contain two distinct types of neurones displaying immunoreactivity for substance P (neurokinin-1) receptor (SPR). Large and medium-sized SPR-immunoreactive neurones, both with aspiny dendrites, were fairly uniformly distributed in the striatum of humans and squirrel monkeys. In humans the proportions of large and medium-sized SPR-positive neurones were 57.2% and 42.8% in putamen, compared with 51.9% and 48.1% in caudate nucleus. These findings suggest that substance P exerts its local influence not only on large cholinergic neurones, as commonly believed, but also on a subset of medium-sized interneurones in the striatum of human and non-human primates.

Calcium-binding proteins in primate basal ganglia.

This paper describes the distribution of the calcium-binding proteins calbindin-D28k. Parvalbumin and calretinin in primate basal ganglia. The data derive from immunocytochemical studies undertaken in squirrel monkeys (Saimiri sciureus) and in normal human individuals. In the striatum, calbindin labels medium-sized spiny projection neurons whereas parvalbumin and calretinin mark two separate classes of aspiny interneurons. The striatal matrix compartment is markedly enriched with calbindin while striatal patches (striosomes) display a calretinin-rich neuropil. In the pallidum, virtually all neurons contain parvalbumin but none express calbindin. Calretinin occurs only in a small subpopulation of both large and small pallidal neurons. In the subthalamic nucleus, there exists a multitude of parvalbumun-positive cells and fibers but the number of calretinin and calbindin-positive neuronal elements is small. In the substantia nigra/ventral tegmental area complex, calbindin and calretinin occur principally in dopaminergic neurons of the dorsal tier of the pars compacta and in those of the ventral tegmental area. Parvalbumin is strictly confined to the GABAergic neurons of the pars reticulata and lateralis. Calbindin-rich fibers abound in the pars reticulata and lateralis, while calretinin-positive axons are confined to the pars compacta. These results indicate that calbindin and parvalbumin are distributed according to a strikingly complementary pattern in primate basal ganglia. Calretinin is less ubiquitous but occurs in all basal ganglia components where it labels distinct subsets of neurons. Such highly specific patterns of distribution indicate that calbindin, parvalbumin and calretinin may work in synergy within primate basal ganglia.

Calretinin-immunoreactive neurons in the human striatum.

The human striatum contains two types of neurons displaying immunoreactivity for the calcium-binding protein calretinin (CR): (1) large (22, 44 microns>), multipolar neurons with 5-7 long, aspiny and tightly branched dendrites, and (2) medium-sized (9-18 microns), round-to-oval neurons with 2-3 long, varicose and poorly branched dendrites. These CR neurons represent only a small proportion of the total neuronal population and they are heterogeneously distributed in the striatum. The large CR neurons are more numerous in the putamen than in the caudate nucleus, whereas the inverse is true for the medium-sized CR neurons. The ratio of large- to medium-size CR neurons is 1:4 in the putamen compared to 1:6 in the caudate nucleus. The existence of these two distinct subsets of chemospecific striatal neurons suggest that CR may play an important role in the intrinsic organization of the human striatum.