Presence of tau pathology within foetal neural allografts in patients with Huntington's and Parkinson's disease.

Cell replacement has been explored as a therapeutic strategy to repair the brain in patients with Huntington's and Parkinson's disease. Post-mortem evaluations of healthy grafted tissue in such cases have revealed the development of Huntington- or Parkinson-like pathology including mutant huntingtin aggregates and Lewy bodies. An outstanding question remains if tau pathology can also be seen in patients with Huntington's and Parkinson's disease who had received foetal neural allografts. This was addressed by immunohistochemical/immunofluorescent stainings performed on grafted tissue of two Huntington's disease patients, who came to autopsy 9 and 12 years post-transplantation, and two patients with Parkinson's disease who came to autopsy 18 months and 16 years post-transplantation. We show that grafts also contain tau pathology in both types of transplanted patients. In two patients with Huntington's disease, the grafted tissue showed the presence of hyperphosphorylated tau [both AT8 (phospho-tau Ser202 and Thr205) and CP13 (pSer202) immunohistochemical stainings] pathological inclusions, neurofibrillary tangles and neuropil threads. In patients with Parkinson's disease, the grafted tissue was characterized by hyperphosphorylated tau (AT8; immunofluorescent staining) pathological inclusions, neurofibrillary tangles and neuropil threads but only in the patient who came to autopsy 16 years post-transplantation. Abundant tau-related pathology was observed in the cortex and striatum of all cases studied. While the striatum of the grafted Huntington's disease patient revealed an equal amount of 3-repeat and 4-repeat isoforms of tau, the grafted tissue showed elevated 4-repeat isoforms by western blot. This suggests that transplants may have acquired tau pathology from the host brain, although another possibility is that this was due to acceleration of ageing. This finding not only adds to the recent reports that tau pathology is a feature of these neurodegenerative diseases, but also that tau pathology can manifest in healthy neural tissue transplanted into the brains of patients with two distinct neurodegenerative disorders.

Mutant huntingtin is present in neuronal grafts in Huntington disease patients.

OBJECTIVE: Huntington disease (HD) is caused by a genetically encoded pathological protein (mutant huntingtin [mHtt]), which is thought to exert its effects in a cell-autonomous manner. Here, we tested the hypothesis that mHtt is capable of spreading within cerebral tissue by examining genetically unrelated fetal neural allografts within the brains of patients with advancing HD. METHODS: The presence of mHtt aggregates within the grafted tissue was confirmed using 3 different types of microscopy (bright-field, fluorescence, and electron), 2 additional techniques consisting of Western immunoblotting and infrared spectroscopy, and 4 distinct antibodies targeting different epitopes of mHtt aggregates. RESULTS: We describe the presence of mHtt aggregates within intracerebral allografts of striatal tissue in 3 HD patients who received their transplants approximately 1 decade earlier and then died secondary to the progression of their disease. The mHtt(+) aggregates were observed in the extracellular matrix of the transplanted tissue, whereas in the host brain they were seen in neurons, neuropil, extracellular matrix, and blood vessels. INTERPRETATION: This is the first demonstration of the presence of mHtt in genetically normal and unrelated allografted neural tissue transplanted into the brain of affected HD patients. These observations raise questions on protein spread in monogenic neurodegenerative disorders of the central nervous system characterized by the formation of mutant protein oligomers/aggregates.

Cografting of carotid body cells improves the long-term survival, fiber outgrowth and functional effects of grafted dopaminergic neurons.

AIMS: A major limiting factor for cell therapy in Parkinson's disease is that the survival of grafted dopaminergic neurons is very poor, which may be improved by administration of GDNF, for which the carotid body is a good source. MATERIALS & METHODS: Rats with total unilateral dopaminergic denervation were grafted with a cell suspension of rat dopaminergic neuroblasts with or without cell aggregates from the rat carotid body. At 1, 2 and 3 months after grafting, the rats were tested in the cylinder and the rotometer and killed 4 months after grafting. RESULTS: We observed that the survival of dopaminergic neurons and graft-derived dopaminergic innervation were higher in rats that received mixed grafts. Both grafted groups showed complete recovery in the amphetamine-induced rotation test. However, rats with cografts performed significantly better in the cylinder test. CONCLUSION: Cografting of carotid body cells may constitute a useful strategy for cell therapy in Parkinson's disease.

Serotonergic neurons mediate dyskinesia side effects in Parkinson's patients with neural transplants.

Troublesome involuntary movements in the absence of dopaminergic medication, so-called off-medication dyskinesias, are a serious adverse effect of fetal neural grafts that hinders the development of cell-based therapies for Parkinson's disease. The mechanisms underlying these dyskinesias are not well understood, and it is not known whether they are the same as in the dyskinesias induced by l-dopa treatment. Using in vivo brain imaging, we show excessive serotonergic innervation in the grafted striatum of two patients with Parkinson's disease, who had exhibited major motor recovery after transplantation with dopamine-rich fetal mesencephalic tissue but had later developed off-medication dyskinesias. The dyskinesias were markedly attenuated by systemic administration of a serotonin [5-hydroxytryptamine (5-HT)] receptor (5-HT(1A)) agonist, which dampens transmitter release from serotonergic neurons, indicating that the dyskinesias were caused by the serotonergic hyperinnervation. Our observations suggest strategies for avoiding and treating graft-induced dyskinesias that result from cell therapies for Parkinson's disease with fetal tissue or stem cells.

Human fetal striatal transplantation in huntington's disease: a refinement of the stereotactic procedure.

BACKGROUND: Human fetal striatal transplantation (HFST) is an experimental stereotactic intervention in the treatment of Huntington's disease (HD). This procedure has proved feasible, safe, well tolerated and it offers a potential strategy for brain repair in HD patients. Target areas are the nucleus caudatus caput (NCc) and the precommissural and postcommissural putamen (Pu). A suboptimal spatial distribution of grafts was frequently reported, especially for the postcommissural Pu, because of striatal atrophy and the concurrent ventricular frontal horn enlargement. An improvement of the stereotactic procedure aimed to optimize the intrastriatal placement of grafts is therefore considered a timely issue. METHODS: Eight consecutive HD patients underwent bilateral HFST. For the first 6 procedures (first group) we performed both caudate and putaminal tracks through a single frontal entry point. For the following 10 procedures (second group), we adopted two completely distinct routes, with two separate entry points, for NCc and Pu tracks. The average number of stereotactic tracks and the average infused volume of tissue suspension were compared between the two groups. RESULTS: The average number of putaminal tracks and the average infused volume of suspension were significantly higher in the second group. CONCLUSION: Adopting two separate routes for caudate and putaminal trajectories allowed us to achieve a larger amount of fetal tissue deposits and a better spatial distribution of grafts.

Transplantation of rat neural stem cells reduces stereotypic behaviors in rats after intrastriatal microinfusion of Tourette syndrome sera.

Tourette syndrome (TS) is a heterogenous neuropsychiatric disorder. In most cases, tics are self-limited or can be treated by behavioral or pharmacological therapy. However, for some individuals, tics can cause lifelong impairment and life-threatening symptoms, which are intractable to traditional treatment. Neural stem cell (NSC) is a potential tool to treat certain neurological diseases. In this study, we proposed to use neural stem cell transplantation as a novel therapy to treat TS and discussed its efficacy. Wistar rats were microinfused with TS sera into the striatum followed by the transplantation of NSCs or vehicle at the infusion site. The sera of the TS patients were identified to have enriched antineural antibodies. Prior to grafting, rat embryonic NSCs were co-cultured with 5-bromodeoxyuridine (Brdu) for 24 h. Stereotypic behaviors were counted at 1, 7, 14 and 21 days after transplantation of NSCs. Morphological analyses revealed that NSCs survived and differentiated into neurons and astrocytes in the striatum 3 weeks after grafting. To sum it up, rat embryonic neural stem cell grafts survived and differentiated in the striatum of TS rat may help relieve stereotypic behaviors of the host. Our results suggest that transplantation of NSCs intrastriatum may have therapeutic potential for TS.

The corridor task: striatal lesion effects and graft-mediated recovery in a model of Huntington's disease.

Experimental validation of cell replacement therapy as a treatment of neurodegenerative diseases requires the demonstration of graft-mediated behavioural recovery. The Corridor task proved to be simple and efficient to conduct with a robust ipsilateral retrieval bias in our rodent Huntington's disease model. The Corridor task is a viable behavioural option, particularly to non-specialised laboratories, for the evaluation of lateralised striatal damage and the probing of alternative therapeutic strategies, including transplantation.

Stem cell transplantation for Huntington's disease.

By way of commentary on a recent report that transplanted adult neural progenitor cells can alleviate functional deficits in a rat lesion model of Huntington's disease [Vazey, E.M., Chen, K., Hughes, S.M., Connor, B., 2006. Transplanted adult neural progenitor cells survive, differentiate and reduce motor function impairment in a rodent model of Huntington's disease. Exp. Neurol. 199, 384-396], we review the current status of the field exploring the use of stem cells, progenitor cells and immortalised cell lines to repair the lesioned striatum in animal models of the human disease. A remarkably rich range of alternative cell types have been used in various animal models, several of which exhibit cell survival and incorporation in the host brain, leading to subsequent functional recovery. In comparing the alternatives with the 'gold standard' currently offered by primary tissue grafts, key issues turn out to be: cell survival, differentiation prior to and following implantation into striatal-like phenotypes, integration and connectivity with the host brain, the nature of the electrophysiological, motor and cognitive tests used to assess functional repair, and the mechanisms by which the grafts exert their function. Although none of the alternatives yet has the capacity to match primary fetal tissues for functional repair, that standard is itself limited, and the long term goal must be not just to match but to surpass present capabilities in order to achieve fully functional reconstruction reliably, flexibly, and on demand.

Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat.

Repeated systemic administration of low doses of kainic acid (KA) induces spontaneous convulsive seizures [Hellier JL, Patrylo PR, Buckmaster PS, Dudek FE. Recurrent spontaneous motor seizures after repeated low-dose systemic treatment with kainate: assessment of a rat model of temporal lobe epilepsy. Epilepsy Res 1998;31:73-84]. In this study, male Sprague-Dawley animals received intranigral transplants of a control cell line M213-2O, or a cell line transfected with human GAD67 cDNA (M213-2O CL4) [Conejero-Goldberg C, Tornatore C, Abi-Saab W, Monaco MC, Dillon-Carter O, Vawter M, et al. Transduction of human GAD67 cDNA into immortalized striatal cell lines using an Epstein-Barr virus-based plasmid vector increases GABA content. Exp Neurol 2000;161:453-61], or no transplant. Eight weeks after transplantation surgery, KA was administered (5 mg/kg/h) until animals reached stage V seizures as described by Racine [Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281-94]. The group transplanted with CL4 required a larger dose of KA and a longer latency to reach a stage V seizure. In addition, this group exhibited significantly fewer stage III and IV seizures. These results indicate that intranigral transplants of a GABA-producing cell line can decrease the number of kainic acid-induced seizures.

Integrating fetal neural transplants into a therapeutic strategy: the example of Huntington's disease.

Fetal neural transplants have become clinically relevant over the past 15 years for two major neurodegenerative diseases, namely Parkinson's disease and Huntington's disease. It is therefore timely to consider how this neurosurgical procedure can integrate the therapeutic armamentarium, what can be expected of it, and what cannot. We use here the example of Huntington's disease to show what fetal neural transplants may uniquely offer for that disease. Up to very recent times, Huntington's disease has been one special example of those neurodegenerative diseases against which neurologists feel totally helpless. This has all changed today and, although results are essentially still to come, one can foresee the mobilization of very large scientific and medical forces against this disease, with definite steps forward in terms of physiopathology and a better view of the therapeutic challenges. While defining the role that fetal neural transplantation may play in meeting these challenges, we also try to show rationales and developments for all types of treatments attempted or suggested so far, as well as their limits and, when relevant, informative failures. The date of writing this review needs to be noted, because the rapid accumulation of data on molecular mechanisms of Huntington's disease pathogenesis and the increasing numbers of clinical trials do not allow much time for the ink of a review to dry.

Cell therapy in Huntington's disease.

Huntington's disease is an autosomal dominant genetic disease, which results in progressive neuronal degeneration in the neostriatum and neocortex, and associated functional impairments in motor, cognitive, and psychiatric domains. Although the genetic mutation is identified, involving an abnormal CAG expansion within the htt gene on chromosome 4, the mechanism by which this leads to neuronal cell death and the question of why striatal neurones are targeted both remain unknown. Thus, in addition to the search for molecular and genetic strategies to inhibit development of the disease, we still need to identify effective strategies for cellular repair in affected individuals. Aspects of the human neuropathology can be well modeled by excitotoxic or metabolic lesions in experimental animals, and in transgenic mice carrying the htt mutation, providing the basis for testing alternative therapeutic strategies. The rationale and efficacy of alternative cell therapies are reviewed, including transplantation repair with embryonic striatal tissues, expansion and differentiation of striatal-like cells from stem cells, and in vivo and ex vivo gene therapy for delivery of neuroprotective growth factor molecules. Pilot and experimental clinical trials of several approaches are now also underway, and the alternative strategies are compared.

Staging and preparation of human fetal striatal tissue for neural transplantation in Huntington's disease.

Transplantation of human fetal central nervous system tissue has been shown to be of benefit in Parkinson's disease, and is currently being explored as a therapeutic option in Huntington's disease. The success of a neural transplant is dependent on a number of factors, including the requirement that donor cells are harvested within a given developmental window and that the cell preparation protocols take account of the biological parameters identified in animal models. Although many of the criteria necessary for a successful neural transplant have been defined in animal models, ultimately they must be validated in human studies, and some issues can only ever be addressed in human studies. Furthermore, because neural transplantation of human fetal tissue is limited to small numbers of patients in any one surgical center, largely due to practical constraints, it is crucial that tissue preparation protocols are clearly defined and reproducible, so that (i) multicenter trials are possible and are based on consistent tissue preparation parameters, and (ii) results between centers can be meaningfully analyzed. Here we describe the preparation of human fetal striatum for neural transplantation in Huntington's disease, and report on the validation of a method for estimating the developmental stage of the fetus based on direct morphometric measurements of the embryonic tissue.

Long-term hibernation of human fetal striatal tissue does not adversely affect its differentiation in vitro or graft survival: implications for clinical trials in Huntington's disease.

Transplantation of human fetal CNS tissue is a promising therapy for neurodegenerative conditions such as Huntington's disease (HD), but its widespread adoption is limited by restricted tissue availability. One method of overcoming this problem would be to store the tissue in hibernation medium, an approach that we reported previously for human fetal striatal tissue stored for up to 24 h. We now demonstrate the feasibility of storing such tissue for up to 8 days in hibernation medium. When either fresh or 8-day hibernated striatal cells were cultured under standard conditions for 4 days, the proportion of DARPP-32-positive neurons did not differ significantly, although the total number of cells was significantly less from tissue that had been hibernated. Six weeks after transplantation into cyclosporin A-immunosuppressed unilateral quinolinic acid-lesioned rats, there was no significant difference between fresh and hibernated grafts, both in terms of graft volume and extent of striatal phenotypic markers. This study therefore clearly demonstrates that hibernation of human fetal striatal tissue for up to 8 days is not deleterious to its differentiation in culture or survival following transplantation, and is therefore an appropriate method of storage for this tissue.

Motor training effects on recovery of function after striatal lesions and striatal grafts.

Environment, training, and experience can influence plasticity and recovery of function after brain damage. However, it is less well known whether, and how, such factors influence the growth, integration, and functional recovery provided by neural grafts placed within the brain. To explore this process, rats were pretrained on the skilled staircase test, then lesioned unilaterally in the lateral dorsal striatum with quinolinic acid. Half of the animals were given suspension grafts prepared from E15 whole ganglionic eminence implanted into the lesioned striatum. For the following 5 months, half of the animals in each group were trained daily in a bilateral manual dexterity task. Then, 23 weeks after surgery, all animals were retested on the staircase test. The grafts promoted recovery in the reaching task, irrespective of the additional dexterity training, and within the trained group recovery was proportional to the volume of the striatal-like tissue in the graft, suggesting that training influenced the pattern of graft-induced functional recovery. The additional training also benefited the rats with lesions alone, raising their performance close to level of the grafted groups. In separate tests of rotation, the grafts reduced drug-induced ipsilateral turning in response to both amphetamine and apomorphine, an effect that was greater in the grafted rats given extra training. The results suggest that both nonspecific motor training and cell transplantation can contribute to recovery of lost function in tests of spontaneous and skilled lateralized motor function after striatal damage, and that these two factors interact in a task-specific manner.

[Criteria of efficiency of transplantation of embryonic nervous tissue preparations in rats with 6-OHDA-impaired dopaminergic nigrostriatal system]. / Kriterii éffektivnosti transplantatsii preparatov émbrional'noi nervnoi tkani u krys s povrezhdeniem dofaminergicheskoi nigrostriarnoi sistemy 6-gidrooksidofaminom (6-OHDA).

Effectiveness of transplantation of cells from embryonal nervous tissue of the ventral mesencephalon (VM ENT) and striatum (STR ENT) by apomorphin-induced motor asymmetry (APO-test), consolidation of the transplant (the degree of glyal reaction and amount of dopaminergic neurons) and blood serum levels of GFAP was studied for 3 months in Wistar rats with 6-OHDA-impaired dopaminergic nigrostriatal system. Marked therapeutic effectiveness was registered in VM ENT transplantation in the denervated striatum and in combined transplantation of VM ENT into the lateral cerebral ventricle simultaneously with STR ENT transplantation in the striatum. Separate transplantation of VM ENT in the lateral ventricle and STR ENT in the striatum had no positive effect on recovery of the dopaminergic nigrostriatal system. A correlation was found between the degree of glial reaction of ENT transplants, severity of rotation asymmetry and serum levels of gliofibrillary protein (GFAP). GFAP in the serum for lifetime assessment of transplant consolidation and prognosis of neurotransplantation efficiency was assayed.

Neural cells from primary human striatal xenografts migrate extensively in the adult rat CNS.

Primary neural cells do not appear to migrate significantly following transplantation into the adult rodent CNS, which is in contrast to expanded neural precursor cells where migration is well-documented. However, most transplant studies of primary neural tissue have been performed in an allograft situation in which it is difficult to identify graft-derived cells. We have, therefore, used a xenograft paradigm to investigate the potential for cells derived from grafts of primary human fetal striatal tissue (gestational age of 66-72 days) to migrate following intrastriatal transplantation in an athymic adult rat model of Huntington's disease. The use of an antibody specific to human nuclear antigen enabled clear identification of graft-derived cells within the host brain, and specific neural phenotypes were determined using human-specific tau for neurons, glial fibrillary acidic protein for mature astrocytes and Ki67 for proliferative cells. At 6 weeks, the graft mass was very dense with a high proliferative index, few cells had migrated away from the graft, and the cells that had differentiated both within and away from the graft were mainly neurons. In contrast, at 6 months, the graft core was dispersed significantly more and a large number of graft-derived cells had migrated throughout the brain as far rostral as the olfactory bulb and as caudal as the substantia nigra. Cells had differentiated into both neurons and astrocytes and the level of proliferation was significantly lower within the graft. These results demonstrate that primary neural xenografts contain proliferative cells that possess the ability to migrate and differentiate into both neurons and astrocytes, and suggest that these cells could contribute to normal graft function. This property may be a consequence of the xenograft situation and could potentially be exploited to provide the opportunity to target regions of distant pathology in neurodegenerative diseases using xenotransplantation of embryonic neural tissue.

Induction of tyrosine hydroxylase expression in rat fetal striatal precursor cells following transplantation.

Differentiation of neural stem cells into tyrosine hydroxylase (TH)-expressing cells was studied by cell transplantation into the various brain regions of rats that had received 6-hydroxydopamine lesion of the nigrostriatal pathway. Approximately 13.6-16.1% of survived precursor cells acquired neuronal-like features by expressing the neuronal marker doublecortin. Similarly, 20.7-25.7% of survived precursor cells differentiated into astrocytes following transplantation. Immunohistochemical analysis revealed that a fraction of the grafted precursor cells in the anterior part of the medial forebrain bundle (MFB), but not in the striatum and the substantia nigra of the lesioned side, showed strong TH immunoreactivity. This suggests that MFB is more permissive for induction of TH to the striatal precursor cells in vivo. The results further exemplify the potential of neural stem cells and the property of site-specific differentiation when the cells were transplanted to the dopaminergic system of the adult brain.

Simultaneous intrastriatal and intranigral dopaminergic grafts in the parkinsonian rat model: role of the intranigral graft.

The current transplantation strategy in experimental and clinical Parkinson's disease (PD) has been to place nigral dopaminergic grafts not in their ontogenic site (substantia nigra) but in their target area (striatum). Although intrastriatal dopaminergic grafts are capable of reinnervating the striatum, they fail to reinnervate the nigra, which may be an important factor limiting the efficacy of fetal tissue transplantation in parkinsonian patients. We have previously shown that simultaneous intrastriatal and intranigral dopaminergic grafts (double grafts) may provide a more complete restoration of the nigrostriatal circuitry (Mendez et al. [1996] J Neurosci 16:7216-7227; Mendez and Hong [1997] Brain Res 778:194-205). In the present study, we investigated the contribution of the intranigral graft to functional recovery in double-grafted hemiparkinsonian rats. Twenty Wistar rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway were divided into two groups and received either double grafts (n = 10) or intrastriatal grafts alone (n = 10). Following transplantation, both intrastriatally and double-grafted animals had a significant decrease in rotational behavior. However, only animals with double grafts exhibited a significant increase in contralateral adjusting step performance. The intranigral graft was subsequently lesioned by a second 6-OHDA injection. Following the second lesion, animals with double grafts exhibited a significant reversal of rotational behavior and a 51% reduction in contralateral adjusting step performance. The reversal in functional recovery correlated with a significant loss of intranigral grafted neurons. These results suggest that the intranigral graft has an important role in the functional recovery of double-grafted animals. Restoration of dopaminergic innervation to both the nigra and the striatum may be crucial for optimizing graft efficacy and may be a superior strategy in neural transplantation for PD.

Aspects of PET imaging relevant to the assessment of striatal transplantation in Huntington's disease.

Proper assessment of outcome in clinical trials of neural transplantation requires both biochemical and imaging indices of graft survival, and behavioural and physiological indices of graft function. For transplantation in Huntington's disease, a variety of ligands that are selective for striatal degeneration and graft-derived replacement are available, notably ligands of dopaminergic receptors on striatal neurons. However, the validity of such ligands is potentially compromised by adjunctive drug therapies (e.g. neuroleptics) given to patients in the course of normal clinical care. We review the present state of experimental and clinical understanding of the selectivity of available ligands for striatal imaging, their interaction with other drug treatments, and strategies for refining valid assessment protocols in patients.