Jeffrey H. Kordower

A Double-blind Controlled Trial of Bilateral Fetal Nigral Transplantation in Parkinson's Disease

Thirty‐four patients with advanced Parkinsons disease participated in a prospective 24‐month double‐blind, placebo‐controlled trial of fetal nigral transplantation. Patients were randomized to receive bilateral transplantation with one or four donors per side or a placebo procedure. The primary end point was change between baseline and final visits in motor component of the Unified Parkinsons Disease Rating Scale in the practically defined off state. There was no significant overall treatment effect (p = 0.244). Patients in the placebo and one‐donor groups deteriorated by 9.4 ± 4.25 and 3.5 ± 4.23 points, respectively, whereas those in the four‐donor group improved by 0.72 ± 4.05 points. Pairwise comparisons were not significant, although the four‐donor versus placebo groups yielded a p value of 0.096. Stratification based on disease severity showed a treatment effect in milder patients (p = 0.006). Striatal fluorodopa uptake was significantly increased after transplantation in both groups and robust survival of dopamine neurons was observed at postmortem examination. Fifty‐six percent of transplanted patients developed dyskinesia that persisted after overnight withdrawal of dopaminergic medication (“off”‐medication dyskinesia). Fetal nigral transplantation currently cannot be recommended as a therapy for PD based on these results.Ann Neurol 2003;54:403–414

Lewy body–like pathology in long-term embryonic nigral transplants in Parkinson's disease

Fourteen years after transplantation into the striatum of an individual with Parkinsons disease, grafted nigral neurons were found to have Lewy body–like inclusions that stained positively for α-synuclein and ubiquitin and to have reduced immunostaining for dopamine transporter. These pathological changes suggest that Parkinsons disease is an ongoing process that can affect grafted cells in the striatum in a manner similar to host dopamine neurons in the substantia nigra. These findings have implications for cell-based therapies and for understanding the cause of Parkinsons disease.

Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease

Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinsons disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinsons disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinsons disease.Human pluripotent stem cells (PSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of PSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However, the effective use of PSCs for cell therapy has lagged behind. Whereas mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons from human PSCs generally show poor in vivo performance. There are also considerable safety concerns for PSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor-plate-based strategy for the derivation of human DA neurons that efficiently engraft in vivo, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor-plate precursors are derived from PSCs 11 days after exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signalling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of PSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in Parkinson’s disease models using three host species. Long-term engraftment in 6-hydroxy-dopamine-lesioned mice and rats demonstrates robust survival of midbrain DA neurons derived from human embryonic stem (ES) cells, complete restoration of amphetamine-induced rotation behaviour and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models indicate promise for the development of cell-based therapies in Parkinson’s disease.

A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.

Neuropathological Evidence of Graft Survival and Striatal Reinnervation after the Transplantation of Fetal Mesencephalic Tissue in a Patient with Parkinson's Disease

BACKGROUND Trials are under way to determine whether fetal nigral grafts can improve motor function in patients with Parkinsons disease. Some studies use fluorodopa uptake on positron-emission tomography (PET) as a marker of graft viability, but fluorodopa uptake does not distinguish between host and grafted neurons. There has been no direct evidence that grafts of fetal tissue can survive and innervate the striatum. METHODS We studied a 59-year-old man with advanced Parkinsons disease who received bilateral grafts of fetal ventral mesencephalic tissue in the postcommissural putamen. The tissue came from seven embryos between 6 1/2 and 9 weeks after conception. The patient died 18 months later from a massive pulmonary embolism. The brain was studied with the use of tyrosine hydroxylase immunohistochemical methods. RESULTS After transplantation, the patient had sustained improvement in motor function and a progressive increase in fluorodopa uptake in the putamen on PET scanning. On examination of the brain, each of the large grafts appeared to be viable. Each was integrated into the host striatum and contained dense clusters of dopaminergic neurons. Processes from these neurons had grown out of the grafts and provided extensive dopaminergic reinnervation to the striatum in a patch-matrix pattern. Ungrafted regions of the putamen showed sparse dopaminergic innervation. We could not identify any sprouting of host dopaminergic processes. CONCLUSIONS Grafts of fetal mesencephalic tissue can survive for a long period in the human brain and restore dopaminergic innervation to the striatum in patients with Parkinsons disease. In the patient we studied, clinical improvement and enhanced fluorodopa with uptake on PET scanning were associated the survival of the grafts and dopaminergic reinnervation of the striatum.

Upregulation of choline acetyltransferase activity in hippocampus and frontal cortex of elderly subjects with mild cognitive impairment

In Alzheimers disease (AD), loss of cortical and hippocampal choline acetyltransferase (ChAT) activity has been correlated with dementia severity and disease duration, and it forms the basis for current therapies. However, the extent to which reductions in ChAT activity are associated with early cognitive decline has not been well established. We quantified ChAT activity in the hippocampus and four cortical regions (superior frontal, inferior parietal, superior temporal, and anterior cingulate) of 58 individuals diagnosed with no cognitive impairment (NCI; n = 26; mean age 81.4 ± 7.3 years), mild cognitive impairment (MCI; n = 18; mean age 84.5 ± 5.7), or mild AD (n =14; mean age 86.3 ± 6.6). Inferior parietal cortex ChAT activity was also assessed in 12 subjects with end‐stage AD (mean age 81.4 ± 4.3 years) and compared to inferior parietal cortex ChAT levels of the other three groups. Only the end‐stage AD group had ChAT levels reduced below normal. In individuals with MCI and mild AD, ChAT activity was unchanged in the inferior parietal, superior temporal, and anterior cingulate cortices compared to NCI. In contrast, ChAT activity in the superior frontal cortex was significantly elevated above normal controls in MCI subjects, whereas the mild AD group was not different from NCI or MCI. Hippocampal ChAT activity was significantly higher in MCI subjects than in either NCI or AD. Our results suggest that cognitive deficits in MCI and early AD are not associated with the loss of ChAT and occur despite regionally specific upregulation. Thus, the earliest cognitive deficits in AD involve brain changes other than simply cholinergic system loss. Of importance, the cholinergic system is capable of compensatory responses during the early stage of dementia. The upregulation in frontal cortex and hippocampal ChAT activity could be an important factor in preventing the transition of MCI subjects to AD.

Missing pieces in the Parkinson's disease puzzle

Parkinsons disease is a neurodegenerative process characterized by numerous motor and nonmotor clinical manifestations for which effective, mechanism-based treatments remain elusive. Here we discuss a series of critical issues that we think researchers need to address to stand a better chance of solving the different challenges posed by this pathology.

Gene delivery of AAV2-neurturin for Parkinson's disease: a double-blind, randomised, controlled trial

BACKGROUND In an open-label phase 1 trial, gene delivery of the trophic factor neurturin via an adeno-associated type-2 vector (AAV2) was well tolerated and seemed to improve motor function in patients with advanced Parkinsons disease. We aimed to assess the safety and efficacy of AAV2-neurturin in a double-blind, phase 2 randomised trial. METHODS We did a multicentre, double-blind, sham-surgery controlled trial in patients with advanced Parkinsons disease. Patients were randomly assigned (2:1) by a central, computer generated, randomisation code to receive either AAV2-neurturin (5·4 × 10¹¹ vector genomes) injected bilaterally into the putamen or sham surgery. All patients and study personnel with the exception of the neurosurgical team were masked to treatment assignment. The primary endpoint was change from baseline to 12 months in the motor subscore of the unified Parkinsons disease rating scale in the practically-defined off state. All randomly assigned patients who had at least one assessment after baseline were included in the primary analyses. This trial is registered at ClinicalTrials.gov, NCT00400634. RESULTS Between December, 2006, and November, 2008, 58 patients from nine sites in the USA participated in the trial. There was no significant difference in the primary endpoint in patients treated with AAV2-neurturin compared with control individuals (difference -0·31 [SE 2·63], 95% CI -5·58 to 4·97; p=0·91). Serious adverse events occurred in 13 of 38 patients treated with AAV2-neurturin and four of 20 control individuals. Three patients in the AAV2-neurturin group and two in the sham surgery group developed tumours. INTERPRETATION Intraputaminal AAV2-neurturin is not superior to sham surgery when assessed using the UPDRS motor score at 12 months. However, the possibility of a benefit with additional targeting of the substantia nigra and longer term follow-up should be investigated in further studies. FUNDING Ceregene and Michael J Fox Foundation for Parkinsons Research.

Loss and atrophy of layer II entorhinal cortex neurons in elderly people with mild cognitive impairment.

Layer II of the entorhinal cortex contains the cells of origin for the perforant path, plays a critical role in memory processing, and consistently degenerates in end‐stage Alzheimers disease. The extent to which neuron loss in layer II of entorhinal cortex is related to mild cognitive impairment without dementia has not been extensively investigated. We analyzed 29 participants who came to autopsy from our ongoing longitudinal study of aging and dementia composed of religious clergy (Religious Orders Study). All individuals underwent detailed clinical evaluation within 12 months of death and were categorized as having no cognitive impairment (n = 8), mild cognitive impairment (n = 10), or mild or moderate Alzheimers disease (n = 11). Sections through the entorhinal cortex were immunoreacted with an antibody directed against a neuron‐specific nuclear protein (NeuN). Stereological counts of NeuN‐immunoreactive stellate cells, their volume, and the volume of layer II entorhinal cortex were estimated. Cases exhibiting no cognitive impairment averaged 639,625 ± 184,600 layer II stellate neurons in the right entorhinal cortex. Individuals with mild cognitive impairment (63.5%; p < 0.0003) and mild or moderate Alzheimers disease (46.06%; p < 0.0017) displayed significant losses of layer II entorhinal cortex neurons relative to those with no cognitive impairment but not relative to each other (p > 0.33). There was also significant atrophy of layer II entorhinal cortex neurons in individuals with mild cognitive impairment (24.1%) and Alzheimers disease (25.1%). The volume of layer II was also reduced in individuals with mild cognitive impairment (26.5%), with a further reduction in those with Alzheimers disease (46.4%). The loss and atrophy of layer II entorhinal cortex neurons significantly correlated with performance on clinical tests of declarative memory. Atrophy of layer II entorhinal cortex and the neurons within this layer significantly correlated with performance on the Mini Mental Status Examination. These data indicate that atrophy and loss of layer II entorhinal cortex neurons occur in elderly subjects with mild cognitive impairment prior to the onset of dementia and suggests that these changes are not exacerbated in early Alzheimers disease. Ann Neurol 2001;49:202–213

Disease duration and the integrity of the nigrostriatal system in Parkinson’s disease

The pace of nigrostriatal degeneration, both with regards to striatal denervation and loss of melanin and tyrosine hydroxylase-positive neurons, is poorly understood especially early in the Parkinsons disease process. This study investigated the extent of nigrostriatal degeneration in patients with Parkinsons disease at different disease durations from time of diagnosis. Brains of patients with Parkinsons disease (n=28) with post-diagnostic intervals of 1-27 years and normal elderly control subjects (n=9) were examined. Sections of the post-commissural putamen and substantia nigra pars compacta were processed for tyrosine hydroxylase and dopamine transporter immunohistochemistry. The post-commissural putamen was selected due to tissue availability and the fact that dopamine loss in this region is associated with motor disability in Parkinsons disease. Quantitative assessments of putaminal dopaminergic fibre density and stereological estimates of the number of melanin-containing and tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta (both in total and in subregions) were performed by blinded investigators in cases where suitable material was available (n=17). Dopaminergic markers in the dorsal putamen showed a modest loss at 1 year after diagnosis in the single case available for study. There was variable (moderate to marked) loss, at 3 years. At 4 years post-diagnosis and thereafter, there was virtually complete loss of staining in the dorsal putamen with only an occasional abnormal dopaminergic fibre detected. In the substantia nigra pars compacta, there was a 50-90% loss of tyrosine hydroxylase-positive neurons from the earliest time points studied with only marginal additional loss thereafter. There was only a ∼10% loss of melanized neurons in the one case evaluated 1 year post-diagnosis, and variable (30 to 60%) loss during the first several years post-diagnosis with more gradual and subtle loss in the second decade. At all time points, there were more melanin-containing than tyrosine hydroxylase-positive cells. Loss of dopaminergic markers in the dorsal putamen occurs rapidly and is virtually complete by 4 years post-diagnosis. Loss of melanized nigral neurons lags behind the loss of dopamine markers. These findings have important implications for understanding the nature of Parkinsons disease neurodegeneration and for studies of putative neuroprotective/restorative therapies.