Persistent dyskinesias in patients with fetal tissue transplantation for Parkinson disease.

Cell transplants are being developed for patients with Parkinson disease (PD) who have insufficient benefit with standard medical treatment. We describe the clinical features of five patients who developed persistent dyskinesias after fetal dopaminergic tissue transplantation. All had levodopa-induced dyskinesias preoperatively. We implanted fetal mesencephalic dopaminergic tissue into the putamina bilaterally in 34 patients with advanced PD. They were not immunosuppressed. Five of 34 patients (15%) developed troublesome choreic or dystonic dyskinesias that persisted despite lowering or discontinuing medications. Attempts to treat the involuntary movements with amantadine, clozapine, anticholinergics, dopamine depletors and other medicines had limited success. Metyrosine eliminated dyskinesias but led to the parkinsonian "off" state. Increasing the dose of levodopa worsened the dyskinesias. Three patients required placement of pallidal stimulators, bilaterally in two and unilaterally in one patient who had only contralateral dyskinesias. The two with the bilateral stimulators had improvement in dyskinesias. The patient with the unilateral pallidal stimulator had a substantial reduction of the dyskinesias, but attempts to treat residual "off" symptoms with levodopa were limited by worsening dyskinesias. Although the number of patients developing these persistent dyskinesias was small, these five patients had dramatic improvement after transplant. As a group, they had milder Parkinson signs at baseline and improved to the point of having minimal parkinsonism, with reduction or elimination of levodopa therapy prior to developing persistent dyskinesias. These involuntary movements establish the principle that fetal dopaminergic tissue transplants can mimic the effects of levodopa, not only in reducing bradykinesia, but also in provoking dyskinesias.

Running wheel exercise reduces α-synuclein aggregation and improves motor and cognitive function in a transgenic mouse model of Parkinson's disease.

Exercise has been recommended to improve motor function in Parkinson patients, but its value in altering progression of disease is unknown. In this study, we examined the neuroprotective effects of running wheel exercise in mice. In adult wild-type mice, one week of running wheel activity led to significantly increased DJ-1 protein concentrations in muscle and plasma. In DJ-1 knockout mice, running wheel performance was much slower and Rotarod performance was reduced, suggesting that DJ-1 protein is required for normal motor activity. To see if exercise can prevent abnormal protein deposition and behavioral decline in transgenic animals expressing a mutant human form of α-synuclein in all neurons, we set up running wheels in the cages of pre-symptomatic animals at 12 months old. Activity was monitored for a 3-month period. After 3 months, motor and cognitive performance on the Rotarod and Morris Water Maze were significantly better in running animals compared to control transgenic animals with locked running wheels. Biochemical analysis revealed that running mice had significantly higher DJ-1, Hsp70 and BDNF concentrations and had significantly less α-synuclein aggregation in brain compared to control mice. By contrast, plasma concentrations of α-synuclein were significantly higher in exercising mice compared to control mice. Our results suggest that exercise may slow the progression of Parkinson's disease by preventing abnormal protein aggregation in brain.

Re-Cloning the N27 Dopamine Cell Line to Improve a Cell Culture Model of Parkinson's Disease.

Parkinson's disease is characterized by the death of dopaminergic neurons in the substantia nigra. To understand the molecular mechanisms of the disease, an in vitro model is important. In the 1990s, we used the SV40 large T antigen to immortalize dopaminergic neurons derived from Embryonic Day 14 rat mesencephalon. We selected a clone for its high expression of dopaminergic neuron markers such as tyrosine hydroxylase (TH), and we named it 1RB3AN27 (N27). Because the original N27 cell line has been passaged many times, the line has become a mixture of cell types with highly variable expression of TH. In the current study, we have performed multiple rounds of clonal cultures and have identified a dopaminergic cell clone expressing high levels of TH and the dopamine transporter (DAT). We have named this new clone N27-A. Nearly 100% of N27-A cells express TH, DAT and Tuj1. Western blots have confirmed that N27-A cells have three to four times the levels of TH and DAT compared to the previous mixed population in N27. Further analysis has shown that the new clone expresses the dopamine neuron transcription factors Nurr1, En1, FoxA2 and Pitx3. The N27-A cells express the vesicular monoamine transporter (VMAT2), but do not express dopamine-beta-hydroxylase (DßH), the enzyme responsible for converting dopamine to norepinephrine. Functional analysis has shown that N27-A cells are more sensitive than N27 cells to neurotoxins taken up by the dopamine transporter such as 6-hydroxydopamine and 1-methyl-4-phenylpyridine (MPP+). The DAT inhibitor nomifensine can block MPP+ induced toxicity. The non-selective toxic effects of hydrogen peroxide were similar in both cell lines. The N27-A cells show dopamine release under basal and depolarization conditions. We conclude that the new N27-A clone of the immortalized rat dopaminergic cell line N27 should provide an improved in vitro model for Parkinson's disease research.

DJ-1 Modulates Nuclear Erythroid 2-Related Factor-2-Mediated Protection in Human Primary Alveolar Type II Cells in Smokers.

Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2-related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.

A Novel Hsp90 Inhibitor Activates Compensatory Heat Shock Protein Responses and Autophagy and Alleviates Mutant A53T α-Synuclein Toxicity.

A potential cause of neurodegenerative diseases, including Parkinson's disease (PD), is protein misfolding and aggregation that in turn leads to neurotoxicity. Targeting Hsp90 is an attractive strategy to halt neurodegenerative diseases, and benzoquinone ansamycin (BQA) Hsp90 inhibitors such as geldanamycin (GA) and 17-(allylamino)-17-demethoxygeldanamycin have been shown to be beneficial in mutant A53T α-synuclein PD models. However, current BQA inhibitors result in off-target toxicities via redox cycling and/or arylation of nucleophiles at the C19 position. We developed novel 19-substituted BQA (19BQA) as a means to prevent arylation. In this study, our data demonstrated that 19-phenyl-GA, a lead 19BQA in the GA series, was redox stable and exhibited little toxicity relative to its parent quinone GA in human dopaminergic SH-SY5Y cells as examined by oxygen consumption, trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and apoptosis assays. Meanwhile, 19-phenyl-GA retained the ability to induce autophagy and potentially protective heat shock proteins (HSPs) such as Hsp70 and Hsp27. We found that transduction of A53T, but not wild type (WT) α-synuclein, induced toxicity in SH-SY5Y cells. 19-Phenyl-GA decreased oligomer formation and toxicity of A53T α-synuclein in transduced cells. Mechanistic studies indicated that mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase signaling was activated by A53T but not WT α-synuclein, and 19-phenyl-GA decreased mTOR activation that may be associated with A53T α-synuclein toxicity. In summary, our results indicate that 19BQAs such as 19-phenyl-GA may provide a means to modulate protein-handling systems including HSPs and autophagy, thereby reducing the aggregation and toxicity of proteins such as mutant A53T α-synuclein.

Substantial Differentiation of Human Neural Stem Cells Into Motor Neurons on a Biomimetic Polyurea.

To find the first restorative treatment for spinal cord injury (SCI), researchers have focused on stem cell therapies. However, one obstacle is the lack of an implantable cell scaffold that can support efficient motor neuron (MN) differentiation and proliferation. We aimed to overcome this through the use of an RGD functionalized novel biomimetic polyurea, optimized to encourage efficient differentiation of MNs. Images taken after 14-days showed increased differentiation (∼40%) of hNSCs into MNs as well as increased cell count on the biomimetic polymer compared to PDL-Laminin coating, indicating that the RGD-polyurea provides a favorable microenvironment for hNSC survival, having promising implications for future SCI therapies.

A nerve guidance conduit with topographical and biochemical cues: potential application using human neural stem cells.

Despite major advances in the pathophysiological understanding of peripheral nerve damage, the treatment of nerve injuries still remains an unmet medical need. Nerve guidance conduits present a promising treatment option by providing a growth-permissive environment that 1) promotes neuronal cell survival and axon growth and 2) directs axonal extension. To this end, we designed an electrospun nerve guidance conduit using a blend of polyurea and poly-caprolactone with both biochemical and topographical cues. Biochemical cues were integrated into the conduit by functionalizing the polyurea with RGD to improve cell attachment. Topographical cues that resemble natural nerve tissue were incorporated by introducing intraluminal microchannels aligned with nanofibers. We determined that electrospinning the polymer solution across a two electrode system with dissolvable sucrose fibers produced a polymer conduit with the appropriate biomimetic properties. Human neural stem cells were cultured on the conduit to evaluate its ability to promote neuronal growth and axonal extension. The nerve guidance conduit was shown to enhance cell survival, migration, and guide neurite extension.

Biomimetic poly(serinol hexamethylene urea) for promotion of neurite outgrowth and guidance.

Nerve function recovery is a major technical challenge in the rehabilitation of patients suffering from severe neuropathies. Facilitating functional recovery requires the creation of a growth-permissive environment that directs the extension and myelination of surviving neurons. To this end, an electrospun nanofiber scaffold composed of arginine-glycine-aspartate-modified poly(serinol hexamethylene urea)-blend-poly-ε-caprolactone (PSHU-RGD/PCL) has been employed. Initially, we investigated the cytotoxicity of PSHU in PC12 cell culture. This was followed by functional examinations of PSHU-RGD for cell viability, proliferation, differentiation, and neurite outgrowth, and finally we examined electrospun scaffolds for guided neurite sprouting. MTT proliferation assays indicated no cytotoxic effects of polymer as compared to laminin-coated surfaces. Functional testing revealed PSHU-RGD surfaces to be comparable to the positive control, laminin-coated surface, in neurite outgrowth studies with average neurite lengths of 84.6 µm (laminin), 218.2 µm (PSHU-RGD), 570.2 µm (laminin + NGF), and 958.2 µm (PSHU-RGD + NGF) after two weeks on homogeneously modified surfaces, and 554.8 µm (nonwoven mats) and 1512.3 µm (uniaxially aligned mats) for PSHU-RGD/PCL + NGF scaffolds after one week. We created PSHU functionalized with the tripeptide, RGD, which provided chemical and physical cues to PC12 cell proliferation and differentiation. We expect that PSHU-RGD will be capable of directing and promoting neurite outgrowth in many neuropathy models.

Human sensory neurons derived from induced pluripotent stem cells support varicella-zoster virus infection.

After primary infection, varicella-zoster virus (VZV) establishes latency in neurons of the dorsal root and trigeminal ganglia. Many questions concerning the mechanism of VZV pathogenesis remain unanswered, due in part to the strict host tropism and inconsistent availability of human tissue obtained from autopsies and abortions. The recent development of induced pluripotent stem (iPS) cells provides great potential for the study of many diseases. We previously generated human iPS cells from skin fibroblasts by introducing four reprogramming genes with non-integrating adenovirus. In this study, we developed a novel protocol to generate sensory neurons from iPS cells. Human iPS cells were exposed to small molecule inhibitors for 10 days, which efficiently converted pluripotent cells into neural progenitor cells (NPCs). The NPCs were then exposed for two weeks to growth factors required for their conversion to sensory neurons. The iPS cell-derived sensory neurons were characterized by immunocytochemistry, flow cytometry, RT-qPCR, and electrophysiology. After differentiation, approximately 80% of the total cell population expressed the neuron-specific protein, ßIII-tubulin. Importantly, 15% of the total cell population co-expressed the markers Brn3a and peripherin, indicating that these cells are sensory neurons. These sensory neurons could be infected by both VZV and herpes simplex virus (HSV), a related alphaherpesvirus. Since limited neuronal populations are capable of supporting the entire VZV and HSV life cycles, our iPS-derived sensory neuron model may prove useful for studying alphaherpesvirus latency and reactivation.

Dopamine cell transplantation for Parkinson's disease: the importance of controlled clinical trials.

Transplantation of human fetal dopamine neurons into the brain of Parkinson's disease patients started in the late 1980s, less than 10 years after experiments in rats showed that embryonic dopamine neurons from a narrow window of development are suitable for transplantation. For human transplantation, the critical stage of development is 6 to 8 weeks after conception. Because putamen is the basal ganglia structure most depleted of dopamine in Parkinson's disease and because it is the structure most closely mapped to the motor cortex, it has been the primary target for neurotransplantation. The double blind trial conducted at the University of Colorado, Columbia University, and North Shore University is the first controlled surgical trial performed in the field of neurosurgery. Results have shown that transplants of fetal dopamine neurons can survive transplantation without immunosuppression and without regard to the age of the patients. Transplants improved objective signs of Parkinson's disease to the best effects of L-DOPA seen preoperatively. Placebo surgery produced no clinical changes. In subjects in whom transplants replaced the need for L-DOPA, the implants replicated the preoperative effects of L-DOPA, including dyskinesias in susceptible patients. Our trial has provided the first controlled evidence that dopamine cell transplants can improve the clinical state of patients with Parkinson's disease.

Phenylbutyrate up-regulates the DJ-1 protein and protects neurons in cell culture and in animal models of Parkinson disease.

Parkinson disease is caused by the death of midbrain dopamine neurons from oxidative stress, abnormal protein aggregation, and genetic predisposition. In 2003, Bonifati et al. (23) found that a single amino acid mutation in the DJ-1 protein was associated with early-onset, autosomal recessive Parkinson disease (PARK7). The mutation L166P prevents dimerization that is essential for the antioxidant and gene regulatory activity of the DJ-1 protein. Because low levels of DJ-1 cause Parkinson, we reasoned that overexpression might stop the disease. We found that overexpression of DJ-1 improved tolerance to oxidative stress by selectively up-regulating the rate-limiting step in glutathione synthesis. When we imposed a different metabolic insult, A53T mutant α-synuclein, we found that DJ-1 turned on production of the chaperone protein Hsp-70 without affecting glutathione synthesis. After screening a number of small molecules, we have found that the histone deacetylase inhibitor phenylbutyrate increases DJ-1 expression by 300% in the N27 dopamine cell line and rescues cells from oxidative stress and mutant α-synuclein toxicity. In mice, phenylbutyrate treatment leads to a 260% increase in brain DJ-1 levels and protects dopamine neurons against 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) toxicity. In a transgenic mouse model of diffuse Lewy body disease, long-term administration of phenylbutyrate reduces α-synuclein aggregation in brain and prevents age-related deterioration in motor and cognitive function. We conclude that drugs that up-regulate DJ-1 gene expression may slow the progression of Parkinson disease by moderating oxidative stress and protein aggregation.

Pallidal neuronal discharge in Parkinson's disease following intraputamenal fetal mesencephalic allograft.

BACKGROUND: Human intrastriatal fetal allografts survive over long periods of time in the brains of Parkinson's disease (PD) patients and integrate into host circuitry. However, some grafted patients with a prior history of levodopa-induced dyskinesias have developed off-medication dyskinesias and dystonias following allografting whose mechanism remains poorly understood. The authors present single-unit discharge characteristics in the external and internal globus pallidus (GPe and GPi) in an awake patient with PD undergoing microelectrode-guided surgery for pallidal deep brain stimulation, 10 years following bilateral intraputamenal fetal mesencephalic allografting in an NIH-funded protocol. METHODS: Pallidal single-unit activity at 'rest' and during active movement was evaluated and compared with data sets from 13 PD patients in the 'off-medication' state and from one non-dyskinetic PD patient in the 'on-medication' state. RESULTS AND DISCUSSION: Analysis of firing rate, bursting discharge and oscillatory activity showed that the graft corrected some, but not all, of the abnormalities associated with the off-medication state. Additionally, in the transplanted patient, voluntary hand movement produced a marked reduction in pallidal discharge rate at multiple GPi recording sites, which was not observed during active movement in other patients. These findings are consistent with a persistent effect of transplanted dopamine cells on basal ganglia outflow and suggest a mechanism for the graft-induced dystonic phenotype.

Embryonic stem cells with GFP knocked into the dopamine transporter yield purified dopamine neurons in vitro and from knock-in mice.

Parkinson's disease (PD) is characterized by the selective loss of midbrain dopamine neurons. Neural transplantation with fetal dopamine neurons can be an effective therapy for patients with PD, but recovery of human fetal cells is difficult. Scarcity of tissue has limited clinical application to a small number of research subjects worldwide. Selective differentiation of embryonic stem cells (ESCs) to dopamine neurons could lead to an unlimited supply of cells for expanded clinical transplantation. To facilitate the differentiation and purification of dopamine neurons, the green fluorescent protein (GFP) gene was inserted into the dopamine transporter (DAT) locus in mouse ESCs using homologous recombination. From these DAT-GFP ESCs, dopamine neurons expressing GFP were successfully produced by in vitro differentiation. The DAT-GFP ESCs were used to generate DAT-GFP knock-in mice. We have found that GFP was colocalized with DAT, Pitx3, Engrailed-1, and tyrosine hydroxylase-positive cells in midbrain, hypothalamus, and olfactory bulb but not in noradrenergic cell regions or other ectopic sites. The GFP-positive dopamine neurons could be isolated from embryonic day-15 ventral midbrain by fluorescence activated cell sorting. These purified dopamine neurons survived reculture and expressed tyrosine hydroxylase and DAT when cocultured with mouse astrocytes or striatal cells. Animals homozygous for DAT-GFP were hyperactive because they had no functional DAT protein. These DAT-GFP knock-in ESCs and mice provide unique tools for purifying dopamine neurons to study their physiology, pharmacology, and genetic profiles.

Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells.

Mouse and human fibroblasts have been transformed into induced pluripotent stem (iPS) cells by retroviral transduction or plasmid transfection with four genes. Unfortunately, viral and plasmid DNA incorporation into chromosomes can lead to disruption of gene transcription and malignant transformation. Tumor formation has been found in offspring of mice generated from blastocysts made mosaic with iPS cells. To proceed with iPS cells for human therapy, reprogramming should be done with transient gene expression. Recently, adenoviral vectors have been used to produce mouse iPS cells without viral integration. Here, we report the successful creation of human iPS cells from embryonic fibroblasts using adenoviral vectors expressing c-Myc, Klf4, Oct4, and Sox2. After screening 12 colonies, three stable iPS cell lines were established. Each cell line showed human embryonic stem cell morphology and surface markers. Southern blots and polymerase chain reaction demonstrated that there was no viral DNA integration into iPS cells. Fingerprinting and karyotype analysis confirmed that these iPS cell lines are derived from the parent human fibroblasts. The three human iPS cell lines can differentiate to all three germ layers in vitro, including dopaminergic neurons. After s.c. injection into nonobese diabetic-severe combined immunodeficient mice, each human iPS line produced teratomas within 5 weeks postimplantation. We conclude that adenoviral vectors can reprogram human fibroblasts to pluripotent stem cells for use in individualized cell therapy without the risk for viral or oncogene incorporation.

Noggin enhances dopamine neuron production from human embryonic stem cells and improves behavioral outcome after transplantation into Parkinsonian rats.

Symptoms of Parkinson's disease have been improved by transplantation of fetal dopamine neurons recovered from aborted fetal tissue, but tissue recovery is difficult. Human embryonic stem cells may provide unlimited cells for transplantation if they can be converted to dopamine neurons and survive transplantation into brain. We have found that the bone morphogenic protein antagonist Noggin increased the number of dopamine neurons generated in vitro from human and mouse embryonic stem cells differentiated on mouse PA6 stromal cells. Noggin effects were seen with either early (for mouse, days 0-7, and for human, days 0-9) or continuous treatment. After transplant into cyclosporin-immunosuppressed rats, human dopamine neurons improved apomorphine circling in direct relation to the number of surviving dopamine neurons, which was fivefold greater after Noggin treatment than with control human embryonic stem cell transplants differentiated only on PA6 cells. We conclude that Noggin promotes dopamine neuron differentiation and survival from human and mouse embryonic stem cells. Disclosure of potential conflicts of interest is found at the end of this article.

Transgenic mice overexpressing tyrosine-to-cysteine mutant human alpha-synuclein: a progressive neurodegenerative model of diffuse Lewy body disease.

Abnormal aggregation of human alpha-synuclein in Lewy bodies and Lewy neurites is a pathological hallmark of Parkinson disease and dementia with Lewy bodies. Studies have shown that oxidation and nitration of alpha-synuclein lead to the formation of stable dimers and oligomers through dityrosine cross-linking. Previously we have reported that tyrosine-to-cysteine mutations, particularly at the tyrosine 39 residue (Y39C), significantly enhanced alpha-synuclein fibril formation and neurotoxicity. In the current study, we have generated transgenic mice expressing the Y39C mutant human alpha-synuclein gene controlled by the mouse Thy1 promoter. Mutant human alpha-synuclein was widely expressed in transgenic mouse brain, resulting in 150% overexpression relative to endogenous mouse alpha-synuclein. At age 9-12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15-18 months showed progressive accumulation of human alpha-synuclein oligomers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21-24 months, alpha-synuclein aggregates were further increased, accompanied by severe behavioral deficits. At this age, transgenic mice developed neuropathology, such as Lewy body-like alpha-synuclein and ubiquitin-positive inclusions, phosphorylation at Ser(129) of human alpha-synuclein, and increased apoptotic cell death. In summary, Y39C human alpha-synuclein transgenic mice show age-dependent, progressive neuronal degeneration with motor and cognitive deficits similar to diffuse Lewy body disease. The time course of alpha-synuclein oligomer accumulation coincided with behavioral and pathological changes, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and eventually lead to neuronal death.

Placebo response in Parkinson's disease: comparisons among 11 trials covering medical and surgical interventions.

Placebo-associated improvements have been previously documented in small series of Parkinson's disease (PD) patients. Using a strict definition of placebo-associated improvement, we examined rates and timing of placebo responses to identify patient- and study-based characteristics, predicting positive placebo response in several PD clinical trials. We collected individual patient data from the placebo groups of 11 medical and surgical treatment trials involving PD patients with differing PD severities and placebo-assignment likelihoods. We defined a positive placebo response as > or = 50% improvement in total Unified Parkinson's Disease Rating Scale motor (UPDRSm) score or a decrease by > or = 2 points on at least two UPDRSm items compared to baseline. We calculated positive placebo response rates at early (3-7 weeks), mid (8-18 weeks), and late (23-35 weeks) stages of follow-up. Odds ratios for patient- and study-based characteristics were obtained from a model fitted using generalized estimating equations. There were 858 patients on placebo who met inclusion criteria for analysis. Three studies involved patients without need of symptomatic treatment, two involved patients without motor fluctuations needing symptomatic treatment, and six (three medical and three surgical) involved patients with motor fluctuations. The overall placebo response rate was 16% (range: 0-55%). Patients with higher baseline UPDRSm scores and studies that focused on PD with motor fluctuations, surgical interventions, or those with a higher probability of placebo assignment showed increased odds of positive placebo response. Placebo responses were temporally distributed similarly during early, mid, and late phases of follow-up. Placebo-related improvements occur in most PD clinical trials and are similarly distributed across all 6 months of follow-up. Recognition of factors that impact placebo response rates should be incorporated into individual study designs for PD clinical trials.

DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T alpha-synuclein toxicity.

DJ-1 is the third gene that has been linked to Parkinson disease. Mutations in the DJ-1 gene cause early onset PD with autosomal recessive inheritance. To clarify the mechanism of DJ-1 protection, we have overexpressed the gene in cultured dopaminergic cells that were then subjected to chemical stress. In the rat dopaminergic cell line, N27, and in primary dopamine neurons, overexpression of wild type DJ-1 protected cells from death induced by hydrogen peroxide and 6-hydroxydopamine. Overexpressing the L166P mutant DJ-1 had no protective effect. By contrast, knocking down endogenous DJ-1 with antisense DJ-1 rendered cells more susceptible to oxidative damage. We have found that DJ-1 improves survival by increasing cellular glutathione levels through an increase in the rate-limiting enzyme glutamate cysteine ligase. Blocking glutathione synthesis eliminated the beneficial effect of DJ-1. Protection could be restored by adding exogenous glutathione. Wild type DJ-1 reduced cellular reactive oxygen species and reduced the levels of protein oxidation caused by oxidative stress. By a separate mechanism, overexpressing wild type DJ-1 inhibited the protein aggregation and cytotoxicity usually caused by A53T human alpha-synuclein. Under these circumstances, DJ-1 increased the level of heat shock protein 70 but did not change the glutathione level. Our data indicate that DJ-1 protects dopaminergic neurons from oxidative stress through up-regulation of glutathione synthesis and from the toxic consequences of mutant humanalpha-synuclein through increased expression of heat shock protein 70. We conclude that DJ-1 has multiple specific mechanisms for protecting dopamine neurons from cell death.

Nectin-1/HveC Mediates herpes simplex virus type 1 entry into primary human sensory neurons and fibroblasts.

Immunocytochemistry detects nectin-1/HveC, nectin-2/HveB, and HVEM/HveA on the surface of sensory neurons and fibroblasts grown as primary cultures from human dorsal root ganglia. Viral entry into these cultured cells was assayed by infection with a recombinant herpes simplex virus type 1 (HSV-1) expressing green fluorescent protein. Soluble, truncated nectin-1 polypeptide, as well as polyclonal and monoclonal antibodies against nectin-1, inhibited infection of neurons, whereas polypeptides and antibodies capable of inhibiting HSV-1 interaction with nectin-2 and herpesvirs entry mediator (HVEM) failed to prevent infection of neuronal cells. These results demonstrate that nectin-1 is the primary receptor for HSV-1 entry into human fetal neurons. Viral entry into fibroblasts was also reduced by soluble nectin-1 but not by soluble HVEM. However, in contrast to the results obtained with neurons, antibodies against receptors failed to inhibit entry into fibroblasts, indicating that unlike neurons, fibroblasts have multiple receptors or mechanisms for HSV-1 entry.