Tobias Piroth

Review: Neurorehabilitation With Neural Transplantation

Cell replacement therapy has been tested clinically in Parkinson’s disease (PD) and Huntington’s disease (HD), epilepsy, spinal cord injury, and stroke. The clinical outcomes have been variable, perhaps partly because of the differing levels of preclinical, basic experimental evidence that was available prior to the trials. The most promising results have been seen in PD trials, with encouraging ones in HD. A common feature of most trials is that they have concentrated on the biological and technical aspects of transplantation without presupposing that the outcomes might be influenced by events after the surgery. The growing evidence of plasticity demonstrated by the brain and grafts in response to environmental and training stimuli such as rehabilitation interventions has been mostly neglected throughout the clinical application of cell therapy. This review suggests that a different approach may be required to maximize recovery: postoperative experiences, including rehabilitation with explicit behavioral retraining, could have marked direct as well as positive secondary effects on the integration and function of grafted cells in the host neural system. The knowledge gained about brain plasticity following brain damage needs to be linked with what we know about promoting intrinsic recovery processes and how this can boost neurobiological and surgical strategies for repair at the clinical level. With proof of principle now established, a rich area for innovative research with profound therapeutic application is open for investigation.

Efficient expansion and dopaminergic differentiation of human fetal ventral midbrain neural stem cells by midbrain morphogens

Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinsons disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach.

One-pass deep brain stimulation of dentato-rubro-thalamic tract and subthalamic nucleus for tremor-dominant or equivalent type Parkinson's disease.

BackgroundRefractory tremor in tremor-dominant (TD) or equivalent-type (EQT) idiopathic Parkinson’s syndrome (IPS) poses the challenge of choosing the best target region to for deep brain stimulation (DBS). While the subthalamic nucleus is typically chosen in younger patients as the target for dopamine-responsive motor symptoms, it is more complicated if tremor does not (fully) respond under trial conditions. In this report, we present the first results from simultaneous bilateral DBS of the DRT (dentato-rubro-thalamic tract) and the subthalamic nucleus (STN) in two elderly patients with EQT and TD IPS and dopamine-refractory tremor.MethodsTwo patients received bilateral octopolar DBS electrodes in the STN additionally traversing the DRT region. Achieved electrode positions were determined with helical CT, overlaid onto DTI tractography data, and compared with clinical data of stimulation response.ResultsBoth patients showed immediate and sustained improvement of their tremor, bilaterally.ConclusionsThe proposed approach appears to be safe and feasible and a combined stimulation of the two target regions was performed tailored to the patients’ symptoms. Clinically, no neuropsychiatric effects were seen. Our pilot data suggest a viable therapeutic option to treat the subgroup of TD and EQT IPS and with tremor as the predominant symptom. A clinical study to further investigate this approach (OPINION: www.clinicaltrials.gov; NCT02288468) is the focus of our ongoing research.

Organization of the human fetal subpallium

The subpallium comprises large parts of the basal ganglia including striatum and globus pallidus. Genes and factors involved in the development of the subpallium have been extensively studied in most vertebrates, including amphibians, birds, and rodents. However, our knowledge on patterning of the human subpallium remains insufficient. Using double fluorescent immunohistochemistry, we investigated the protein distribution of transcription factors involved in patterning of the subventricular zone (SVZ) in the human forebrain at late embryonic development. Furthermore, we compared the development of cortical and striatal precursors between human fetal brain and E14 and E16 fetal rat brains. Our results reveal that DLX2 marks SVZ precursors in the entire subpallium. Individual subpallial subdomains can be identified based on co-expression of DLX2 with either PAX6 or NKX2-1. SVZ precursors in the dorsal LGE and preopto-hypothalamic boundary are characterized by DLX2/PAX6 co-expression, while precursors in the MGE and preoptic region co-express DLX2/NKX2-1. SVZ precursors in the ventral LGE are DLX2(+)/PAX6(-)/NKX2-1(-). In terms of staging comparisons, the development of the corpus striatum in the human fetal brain during late embryonic stages corresponds well with the development of the striatum observed in E14 fetal rat brains. Our study demonstrates that the pattern underlying the development of the subpallium is highly conserved between rodents and humans and suggests a similar function for these factors in human brain development. Moreover, our data directly influence the application of ganglionic eminence derived human tissue for cell therapeutic approaches in neurodegenerative disorders such as Huntingtons disease.

Donor age dependent graft development and recovery in a rat model of Huntington's disease: histological and behavioral analysis.

Neural cell replacement therapy using fetal striatal cells has provided evidence of disease modification in clinical trials in Huntingtons disease (HD) patients, although the results have been inconsistent. One of the contributing factors to the variable outcome could be the different capacity of transplanted cells derived from the primordial striatum to proliferate and maturate into striatal projection neurons. Based on the rodent lesion model of HD, the current study investigated how intrastriatal-striatal grafts from variable aged donors develop in vivo and how they influence functional recovery. Young adult female Sprague-Dawley rats were lesioned unilaterally in the dorso-striatum with quinolinic acid (0.12 M) and transplanted 14 days later with single cell suspension grafts equivalent of one whole ganglionic eminence (WGE) from donors of embryonic developmental age E13, E14, or E15; animals with or without striatal lesion served as controls. All animals were tested on the Cylinder and the Corridor tests, as well as on apomorphine-induced rotation at baseline, post-lesion/pre-grafting, and at 6 and 10 weeks post-grafting. A week prior to perfusion, a sub-group in each grafted group received fluorogold injections into the ipsilateral globus pallidus to study graft efferent projections. In summary, the data demonstrates that the age of the embryonic donor tissue has an impact on both the graft mediated functional recovery, and on the in vivo cellular composition of the striatal transplant. E13 tissue grafts gave the best overall outcome indicating that WGE from different donor ages have different potential to promote functional recovery. Understanding the stages and process in rodent striatal development could improve tissue selection in clinical trials of cell therapy in HD.

Transplantation of human fetal tissue for neurodegenerative diseases: validation of a new protocol for microbiological analysis and bacterial decontamination.

Restorative cell therapy concepts in neurodegenerative diseases are aimed at replacing lost neurons. Despite advances in research on pluripotent stem cells, fetal tissue from routine elective abortions is still regarded as the only safe cell source. Progenitor cells isolated from distinct first-trimester fetal CNS regions have already been used in clinical trials and will be used again in a new multicenter trial funded by the European Union (TRANSEURO). Bacterial contamination of human fetal tissue poses a potential risk of causing infections in the brain of the recipient. Thus, effective methods of microbial decontamination and validation of these methods are required prior to approval of a neurorestorative cell therapy trial. We have developed a protocol consisting of subsequent washing steps at different stages of tissue processing. Efficacy of microbial decontamination was assessed on rat embryonic tissue incubated with high concentrations of defined microbe solutions including representative bacterial and fungal species. Experimental microbial contamination was reduced by several log ranks. Subsequently, we have analyzed the spectrum of microbial contamination and the effect of subsequent washing steps on aborted human fetal tissue; 47.7% of the samples taken during human fetal tissue processing were positive for a microbial contamination, but after washing, no sample exhibited bacterial growth. Our data suggest that human fetal tissue for neural repair can carry microbes of various species, highlighting the need for decontamination procedures. The decontamination protocol described in this report has been shown to be effective as no microbes could be detected at the end of the procedure.

Restoration of the striatal circuitry: from developmental aspects toward clinical applications

In the basal ganglia circuitry, the striatum is a highly complex structure coordinating motor and cognitive functions and it is severely affected in Huntingtons disease (HD) patients. Transplantation of fetal ganglionic eminence (GE) derived precursor cells aims to restore neural circuitry in the degenerated striatum of HD patients. Pre-clinical transplantation in genetic and lesion HD animal models has increased our knowledge of graft vs. host interactions, and clinical studies have been shown to successfully reduce motor and cognitive effects caused by the disease. Investigating the molecular mechanisms of striatal neurogenesis is a key research target, since novel strategies aim on generating striatal neurons by differentiating embryonic stem cells or by reprogramming somatic cells as alternative cell source for neural transplantation.

Novel compound heterozygous synaptojanin-1 mutation causes l-dopa-responsive dystonia-parkinsonism syndrome

impaired. She was unable to read or write. Verbal memory and visuospatial processing were intact. She exhibited symmetric parkinsonism characterized by rigidity, bradykinesia, resting tremor, and impaired postural reflexes (Supporting Information Video is available online). Brain MRI showed asymmetric frontal atrophy (Fig. 1A). She reached a state of akinetic mutism at age 69 and died at age 70. Plasma progranulin levels were 27 ng/ml (normal threshold< 61 ng/ml). GRN sequencing showed a novel heterozygous deletion (g.1642_1645delTGAG) in the 3’ flanking region of exon 7 (IVS7 1 1delTGAG), a highly conserved region in vertebrates (Supplementary Figure). In silico analysis predicted that the wild-type sequence is localized in a donor splice site, which is abolished by the deletion. Microtubule Associated Protein Tau (MAPT) and Chromosome 9 Open Reading Frame 72 (C9ORF72) were negative. At autopsy, the fresh brain weighed 900 grams. Macroscopically, there was severe atrophy of the frontal and temporal lobes. Histologically, there was severe neuronal loss and gliosis involving the neocortex, caudate nucleus, putamen, globus pallidus, hippocampus, dentate nucleus, substantia nigra, and inferior olivary nucleus. Moderate to severe TDP43-immunoreactive neuronal cytoplasmic inclusions as well as dystrophic neurites were observed in the frontal, parietal, and temporal cortices, cingulate gyrus, precuneus, thalamus, subthalamic nucleus, hippocampus, entorhinal cortex, and inferior olivary nucleus (Fig. 1B). These findings are consistent with TDP-43 Type A pathology. Tau immunohistochemistry revealed rare neurofibrillary tangles, tau-immunoreactive neurons and neuropil threads in the putamen, substantia innominata, pyramidal layer of the hippocampus, and entorhinal cortex. Alpha-synuclein was absent. Progranulin expression was absent in the frontal and temporal cortices by semiquantitative real-time polymerase chain reaction expression analysis (Fig. 1C) and strongly reduced in the inferior frontal (216 fold reduction) and temporal lobes (25.7 fold reduction). In the family, the variant (and low progranulin plasma levels) segregated with dementia (Fig. 1D): a sister diagnosed with frontotemporal dementia at 72 years of age, and another with language impairment and gait imbalance at 74 years of age (II:1, II:3). One sister without the GRN mutation developed Parkinson’s disease without cognitive impairment (after 8 years from onset) at 53 years (II:6). Another sister (II:2) had clinical amnestic mild cognitive impairment, but did not convert to dementia at 18 months of follow-up. In conclusion, rapidly progressive nvPPA and parkinsonism was associated in this patient with the novel GRN mutation g.1642_1645delTGAG (IVS711delTGAG). We showed that this variant segregates with dementia in the family and is associated with reduced progranulin plasma levels and TDP-43 histopathology.

Atypical Presentation of Rapid-onset Dystonia-parkinsonism (DYT12) Unresponsive to Deep Brain Stimulation of the Subthalamic Nucleus: STN-DBS in DYT12 patient

Rapid-onset dystonia-parkinsonism (RDP; DYT12; online Mendelian inheritance in man [OMIM] 128235) is a dystoniaplus syndrome characterized by the abrupt manifestation of dystonia, mostly spreading in a rostro-caudal pattern with prominent bulbar symptoms accompanied by parkinsonism features. Onset commonly follows a specific triggering event consisting of physical or psychological stress. The underlying causes are autosomaldominant or de novo mutations in the ATP1A3-gene encoding the alpha-3-subunit of the Na/Ka-ATPase. With about 100 cases described in the literature, it remains a rare disease that, along with its uncommon clinical presentation, may complicate diagnosis. Consequently, little is known about sufficient treatment options.

Bilateral Globus Pallidus Internus Deep Brain Stimulation in a Case of Progressive Dystonia in Mohr-Tranebjaerg Syndrome with Bilateral Cochlear Implants

Introduction A 28‐year‐old man presented with a history of sensorineural deafness since early childhood treated with bilateral cochlear implants (CIs). He showed signs of debilitating dystonia that had been present since puberty. Dystonic symptoms, especially a protrusion of the tongue and bilateral hand tremor, had not responded to botulinum toxin therapy. We diagnosed Mohr‐Tranebjaerg syndrome (MTS). Methods and Material Deep brain stimulation (DBS) of the bilateral globus pallidus internus was performed predominantly with stereotaxic computed tomography angiography guidance under general anesthesia. Electrophysiology was used to identify the target regions and to guide DBS electrode placement. Results In the immediate postoperative course and stimulation, the patient showed marked improvement of facial, extremity, and cervical dystonia. More than 2 years after implantation, his dystonic symptoms had dramatically improved by 82%. Discussion MTS is a rare genetic disorder leading to sensorineural deafness, dystonia, and other symptoms. The use of DBS for the dystonia in MTS was previously described but not in the presence of bilateral CIs. Conclusion DBS in MTS may be a viable option to treat debilitating dystonic symptoms. We describe successful DBS surgery, despite the presence of bilateral CIs, and stimulation therapy over 2 years.