Development and Validation of a Prognostic Model to Predict Overall Survival in Multiple System Atrophy.

Background: Multiple system atrophy (MSA) is a devastating disease characterized by a variable combination of motor and autonomic symptoms. Previous studies identified numerous clinical factors to be associated with shorter survival. Objective: To enable personalized patient counseling, we aimed at developing a risk model of survival based on baseline clinical symptoms. Methods: MSA patients referred to the Movement Disorders Unit in Innsbruck, Austria, between 1999 and 2016 were retrospectively analyzed. Kaplan-Meier curves and multivariate Cox regression analysis with least absolute shrinkage and selection operator penalty for variable selection were performed to identify prognostic factors. A nomogram was developed to estimate the 7 years overall survival probability. The performance of the predictive model was validated and calibrated internally using bootstrap resampling and externally using data from the prospective European MSA Study Group Natural History Study. Results: A total of 210 MSA patients were included in this analysis, of which 124 patients died. The median survival was 7 years. The following clinical variables were found to significantly affect overall survival and were included in the nomogram: age at symptom onset, falls within 3 years of onset, early autonomic failure including orthostatic hypotension and urogenital failure, and lacking levodopa response. The time-dependent area under curve for internal and external validation was >0.7 within the first 7 years of the disease course. The model was well calibrated showing good overlap between predicted and actual survival probability at 7 years. Conclusion: The nomogram is a simple tool to predict survival on an individual basis and may help to improve counseling and treatment of MSA patients.

Safety and Clinical Effects of Mesenchymal Stem Cells Secreting Neurotrophic Factor Transplantation in Patients With Amyotrophic Lateral Sclerosis: Results of Phase 1/2 and 2a Clinical Trials.

IMPORTANCE: Preclinical studies have shown that neurotrophic growth factors (NTFs) extend the survival of motor neurons in amyotrophic lateral sclerosis (ALS) and that the combined delivery of these neurotrophic factors has a strong synergistic effect. We have developed a culture-based method for inducing mesenchymal stem cells (MSCs) to secrete neurotrophic factors. These MSC-NTF cells have been shown to be protective in several animal models of neurodegenerative diseases. OBJECTIVE: To determine the safety and possible clinical efficacy of autologous MSC-NTF cells transplantation in patients with ALS. DESIGN, SETTING, AND PARTICIPANTS: In these open-label proof-of-concept studies, patients with ALS were enrolled between June 2011 and October 2014 at the Hadassah Medical Center in Jerusalem, Israel. All patients were followed up for 3 months before transplantation and 6 months after transplantation. In the phase 1/2 part of the trial, 6 patients with early-stage ALS were injected intramuscularly (IM) and 6 patients with more advanced disease were transplanted intrathecally (IT). In the second stage, a phase 2a dose-escalating study, 14 patients with early-stage ALS received a combined IM and IT transplantation of autologous MSC-NTF cells. INTERVENTIONS: Patients were administered a single dose of MSC-NTF cells. MAIN OUTCOMES AND MEASURES: The primary end points of the studies were safety and tolerability of this cell therapy. Secondary end points included the effects of the treatment on various clinical parameters, such as the ALS Functional Rating Scale-Revised score and the respiratory function. RESULTS: Among the 12 patients in the phase 1/2 trial and the 14 patients in the phase 2a trial aged 20 and 75 years, the treatment was found to be safe and well tolerated over the study follow-up period. Most of the adverse effects were mild and transient, not including any treatment-related serious adverse event. The rate of progression of the forced vital capacity and of the ALS Functional Rating Scale-Revised score in the IT (or IT+IM)-treated patients was reduced (from -5.1% to -1.2%/month percentage predicted forced vital capacity, P < .04 and from -1.2 to 0.6 ALS Functional Rating Scale-Revised points/month, P = .052) during the 6 months following MSC-NTF cell transplantation vs the pretreatment period. Of these patients, 13 (87%) were defined as responders to either ALS Functional Rating Scale-Revised or forced vital capacity, having at least 25% improvement at 6 months after treatment in the slope of progression. CONCLUSIONS AND RELEVANCE: The results suggest that IT and IM administration of MSC-NTF cells in patients with ALS is safe and provide indications of possible clinical benefits, to be confirmed in upcoming clinical trials. TRIAL REGISTRATION: clinicaltrials.gov Identifiers: NCT01051882 and NCT01777646.

Beneficial effect of levodopa therapy on stooped posture in Parkinson's disease.

OBJECTIVE: This study was designated to quantitatively evaluate the effect of levodopa on spinal posture in patients with PD using a computer-assisted handheld SpinalMouse device. METHODS: Prospective case-study involving 48 patients with definite PD. All patients were recruited between September 2011 and September 2013 and included 22 dopa-naïve, evaluated before and 3 months after initiation of treatment, and 26 patients with response fluctuations studied during the "off" and "on" states. The SpinalMouse instrument, a computer-assisted mechanical hand-held device, designed to noninvasively assess the curvature of the spine was guided along the midline of the vertebral column in upright, full flexion, and full extension positions to objectively assess spinal posture. RESULTS: In the dopa-naïve patients, spinal incline in the upright position was 12.4±1.2° before and 7.6±1.3° after treatment; p=0.002. Corresponding area-under-the-curve (AUC) values were 131.7±8.0 cm(2) and 87.1±7.3 cm(2); p<0.0001. In the response fluctuations patients, spinal incline was 13.3±1.3° in the "off" and 9.3±1.2° in the "on" period; p=0.015. Corresponding AUC values were 144.6±9.2 cm(2) and 103.1±8.2 cm(2); p<0.0001. CONCLUSIONS: This is the first study that objectively measured and quantified abnormalities of spinal posture in patients with PD. Findings suggest that levodopa does have a beneficial effect on anterior flexion of the thoracolumbar spine, and thus indicate that the disorder of stooped posture in PD is mediated, at least in part, by dopamine deficiency.

A DJ-1 Based Peptide Attenuates Dopaminergic Degeneration in Mice Models of Parkinson's Disease via Enhancing Nrf2.

Drugs currently used for treating Parkinson's disease patients provide symptomatic relief without altering the neurodegenerative process. Our aim was to examine the possibility of using DJ-1 (PARK7), as a novel therapeutic target for Parkinson's disease. We designed a short peptide, named ND-13. This peptide consists of a 13 amino acids segment of the DJ-1-protein attached to 7 amino acids derived from TAT, a cell penetrating protein. We examined the effects of ND-13 using in vitro and in vivo experimental models of Parkinson's disease. We demonstrated that ND-13 protects cultured cells against oxidative and neurotoxic insults, reduced reactive oxygen species accumulation, activated the protective erythroid-2 related factor 2 system and increased cell survival. ND-13 robustly attenuated dopaminergic system dysfunction and in improved the behavioral outcome in the 6-hydroxydopamine mouse model of Parkinson's disease, both in wild type and in DJ-1 knockout mice. Moreover, ND-13 restored dopamine content in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model. These findings validate DJ-1 as a promising therapeutic target in Parkinson's disease and identify a novel peptide with clinical potential, which may be significant for a broader range of neurological diseases, possibly with an important impact for the neurosciences.

Harnessing neurogenesis for the possible treatment of Parkinson's disease.

The discovery of neurogenesis in the adult brain has created new possibilities for therapeutics in neurodegenerative diseases. Neural precursor cells, which have been found in various parts of the brain, e.g., the subventricular zone (SVZ) and substantia nigra (SN), have promising potential to replace the extensive loss of neurons occurring in neurodegenerative disorders. In Parkinson's disease (PD) the degeneration of nigral dopaminergic neurons and consequently the nigrostriatal pathway, which has been found to innervate proximally to the SVZ, causes motor and cognitive impairments. There is strong evidence that neurogenesis is impaired in PD, which has been related to the nonmotor symptoms of the disease. Recent evidence supports that this impairment in neurogenesis is partially caused by the lack of dopamine in one of the adult neurogenic niches, the SVZ. Thus, restoring the dopaminergic pathway in PD patients may have implications not only for motor function improvement, but for other cognitive and autonomic symptoms. Currently, there are no effective treatments that can stop or reverse the neurodegeneration process in the brain. Here we review the neurogenic process and observed alterations found in PD animal models and postmortem brains of PD patients. Finally, we review several attempts to stimulate the neurogenic process for nigral and/or striatal dopaminergic restoration by transgenic expression, exercise, or cell therapy.

Stem cell grafting in parkinsonism--why, how and when.

Parkinson's disease is a devastating, progressive neurodegenerative disorder that affects the central and peripheral nervous systems. Although recent advancements have led to a better understanding of the disorder, there is currently no long-term disease-modifying strategy. Recently, preclinical data have identified the significant effects of pluripotent stem cell grafting in 6-OHDA and MPTP animal models of motor parkinsonism; there have also been some clinical data in patients with motor parkinsonism. Pluripotent stem cells can nestle in affected organs and can differentiate into a variety of cells, including neural (dopamine producing) cells. Depending on the environment into which they are grafted, these stem cells can also influence immune responses by regulating the activity of B-cells, T-cells, and NK-cells. Pluripotent stem cells can also produce chemotrophins, including BDNF (brain-derived neurotrophic factor), GDNF (glial-derived neurotrophic factor), NGF (nerve growth factor), TGF-ß (transforming growth factor-ß), IGF-1 (insulin-like growth factor 1), NT-3 (neurotrophin 3), and SCF-1 (stem cell factor 1). Influencing these trophic factors can influence plasticity. This article explores the potential of pluripotent stem cells in the treatment of PD. We will explore the utilization of pluripotent stem cells in the immunomodulation of B-cells, T-cells and NK-cells, the transdifferentiation of pluripotent stems cells into DA-cells, and the secretion of trophic factors and its relation to plasticity. We will also cover how best to conduct a clinical trial, which stem cells can be safely used in patients, what are the methods of induction before application, and how to re-apply stem cells in patients by intravasal, intrathecal or intracerebral methods. Finally, we will describe how to objectively record the clinical results.

Perspective: Identification of genetic variants associated with dopaminergic compensatory mechanisms in early Parkinson's disease.

Parkinson's disease (PD) is slowly progressive, and heterogeneity of its severity among individuals may be due to endogenous mechanisms that counterbalance the striatal dopamine loss. In this perspective paper, we introduce a neuroimaging-genetic approach to identify genetic variants, which may contribute to this compensation. First, we briefly review current known potential compensatory mechanisms for premotor and early disease PD, located in the striatum and other brain regions. Then, we claim that a mismatch between mild symptomatic disease, manifested by low motor score on the Unified PD Rating Scale (UPDRS), and extensive Nigro-Striatal (NS) degeneration, manifested by reduced uptake of [(123)I]FP-CIT, is indicative of compensatory processes. If genetic variants are associated with the severity of motor symptoms, while the level of striatal terminals degeneration measured by ligand uptake is taken into account and controlled in the analysis, then these variants may be involved in functional compensatory mechanisms for striatal dopamine deficit. To demonstrate feasibility of this approach, we performed a small "proof of concept" study (candidate gene design) in a sample of 28 Jewish PD patients, and preliminary results are presented.

Knocking out DJ-1 attenuates astrocytes neuroprotection against 6-hydroxydopamine toxicity.

Astrocytes are the most abundant glial cell type in the brain. Impairment in astrocyte functions can critically influence neuronal survival and leads to neurodegeneration. Parkinson's disease (PD) is a common neurodegenerative disorder, characterized by motor dysfunction that results from progressive neuronal loss. Astrocytic dysfunction was demonstrated in human samples and in experimental models of PD. Mutations in DJ-1 (PARK7) leading to loss of functional protein cause familial PD and enhance sensitivity to oxidative insults. Recently, an increase in DJ-1's expression was found in reactive astrocytes in various neurodegenerative disorders. Here we show that lack of DJ-1 attenuates astrocytes' ability to support neuronal cells, thereby leading to accelerated neuronal damage. DJ-1 knockout mice demonstrated increased vulnerability in vivo to 6-hydroxydopamine (6-OHDA) hemiparkinsonian PD model. Astrocytes isolated from DJ-1 knockout mice showed an inferior ability to protect human neuroblastoma cells against 6-OHDA insult both by co-culture and through their conditioned media, as compared to wild-type astrocytes. DJ-1 knockout astrocytes showed blunted ability to increase the expression of cellular protective mechanisms against oxidative stress mediated via Nrf-2 and HO-1 in response to exposure to 6-OHDA. These experiments demonstrated that lack of DJ-1 impairs astrocyte-mediated neuroprotection.

The natural history of multiple system atrophy: a prospective European cohort study.

BACKGROUND: Multiple system atrophy (MSA) is a fatal and still poorly understood degenerative movement disorder that is characterised by autonomic failure, cerebellar ataxia, and parkinsonism in various combinations. Here we present the final analysis of a prospective multicentre study by the European MSA Study Group to investigate the natural history of MSA. METHODS: Patients with a clinical diagnosis of MSA were recruited and followed up clinically for 2 years. Vital status was ascertained 2 years after study completion. Disease progression was assessed using the unified MSA rating scale (UMSARS), a disease-specific questionnaire that enables the semiquantitative rating of autonomic and motor impairment in patients with MSA. Additional rating methods were applied to grade global disease severity, autonomic symptoms, and quality of life. Survival was calculated using a Kaplan-Meier analysis and predictors were identified in a Cox regression model. Group differences were analysed by parametric tests and non-parametric tests as appropriate. Sample size estimates were calculated using a paired two-group t test. FINDINGS: 141 patients with moderately severe disease fulfilled the consensus criteria for MSA. Mean age at symptom onset was 56·2 (SD 8·4) years. Median survival from symptom onset as determined by Kaplan-Meier analysis was 9·8 years (95% CI 8·1-11·4). The parkinsonian variant of MSA (hazard ratio [HR] 2·08, 95% CI 1·09-3·97; p=0·026) and incomplete bladder emptying (HR 2·10, 1·02-4·30; p=0·044) predicted shorter survival. 24-month progression rates of UMSARS activities of daily living, motor examination, and total scores were 49% (9·4 [SD 5·9]), 74% (12·9 [8·5]), and 57% (21·9 [11·9]), respectively, relative to baseline scores. Autonomic symptom scores progressed throughout the follow-up. Shorter symptom duration at baseline (OR 0·68, 0·5-0·9; p=0·006) and absent levodopa response (OR 3·4, 1·1-10·2; p=0·03) predicted rapid UMSARS progression. Sample size estimation showed that an interventional trial with 258 patients (129 per group) would be able to detect a 30% effect size in 1-year UMSARS motor examination decline rates at 80% power. INTERPRETATION: Our prospective dataset provides new insights into the evolution of MSA based on a follow-up period that exceeds that of previous studies. It also represents a useful resource for patient counselling and planning of multicentre trials.

Trial designs used to study neuroprotective therapy in Parkinson's disease.

There have been numerous trials conducted to evaluate putative disease-modifying or neuroprotective treatments in Parkinson's disease. These trials have used several different study designs and outcome measures. Each of these has its own strengths and weaknesses. Confounding all studies is the potential symptomatic benefit that the treatment might have on the features of Parkinson's disease. In addition, patient-related factors such as age of onset and the nature of the dominant symptoms may have important impacts that are often not addressed. Here we provide an overview of the various trial designs that have been used and emphasize the challenges faced in attempting to study neuroprotection in Parkinson's disease and the advances needed before this goal can be successfully achieved.

DJ-1 protects against dopamine toxicity: implications for Parkinson's disease and aging.

Parkinson's disease (PD) is a common age-related neurodegenerative disorder. Dopamine neurotoxicity, mediated through oxidative stress, is implicated in disease pathogenesis. The vesicular monoamine transporter-2 (VMAT2) transfers dopamine into synaptic vesicles preparing it for exocytotic release and preventing its cytoplasmic oxidation. DJ-1 mutations cause early-onset familial PD. Here, we show that DJ-1 protects dopaminergic neurons and controls the vesicular sequestration of dopamine by upregulating VMAT2. Overexpression of DJ-1 protected cells against dopamine toxicity, reduced oxidative stress, and increased VMAT2 expression and function. Reduced DJ-1 levels resulted in opposite effects. Dopamine vesicular sequestration and its release upon depolarization were dependent on DJ-1 levels. Transcriptional regulation of VMAT2 expression by DJ-1 was confirmed by chromatin immunoprecipitation assay. The results were corroborated in vivo using 6-hydroxydopamine hemiparkinsonian mouse model and transgenic DJ-1 knockout mice. Our experimental data point to a novel potential protective function of DJ-1, which could be used as a therapeutic tool.

Intrastriatal transplantation of neurotrophic factor-secreting human mesenchymal stem cells improves motor function and extends survival in R6/2 transgenic mouse model for Huntington's disease.

Stem cell-based treatment for Huntington's disease (HD) is an expanding field of research. Although various stem cells have been shown to be beneficial in vivo, no long standing clinical effect has been demonstrated. To address this issue, we are developing a stem cell-based therapy designed to improve the microenvironment of the diseased tissue via delivery of neurotrophic factors (NTFs). Previously, we established that bone marrow derived human mesenchymal stem cells (MSCs) can be differentiated using medium based cues into NTF-secreting cells (NTF+ cells) that express astrocytic markers. NTF+ cells were shown to alleviate neurodegeneration symptoms in several disease models in vitro and in vivo, including the model for excitotoxicity. In the present study, we explored if the timing of intrastriatal transplantation of hNTF+ cells into the R6/2 transgenic mouse model for HD influences motor function and survival. One hundred thousand cells were transplanted bilaterally into the striatum of immune-suppressed mice at 4.5, 5.5 and 6.5 weeks of age. Contrary to our expectations, early transplantation of NTF+ cells did not improve motor function or overall survival. However, late (6.5 weeks) transplantation resulted in a temporary improvement in motor function and an extension of life span relative to that observed for PBS treated mice. We conclude that late transplantation of NTF+ cells induces a beneficial effect in this transgenic model for HD. Since no transplanted NTF+ cells could be detected in vivo, we suspect that the temporary nature of the beneficial effect is due to poor survival of transplanted cells. In general, we submit that NTF+ cells should be further evaluated for the therapy of HD.

Wnt signaling enhances neurogenesis and improves neurological function after focal ischemic injury.

Stroke potently stimulates cell proliferation in the subventricular zone of the lateral ventricles with subsequent neuroblast migration to the injured striatum and cortex. However, most of the cells do not survive and mature. Extracellular Wnt proteins promote adult neurogenesis in the neurogenic niches. The aim of the study was to examine the efficacy of Wnt signaling on neurogenesis and functional outcome after focal ischemic injury. Lentivirus expressing Wnt3a-HA (LV-Wnt3a-HA) or GFP (LV-GFP) was injected into the striatum or subventricular zone of mice. Five days later, focal ischemic injury was induced by injection of the vasoconstrictor endothelin-1 into the striatum of the same hemisphere. Treatment with LV-Wnt3a-HA into the striatum significantly enhanced functional recovery after ischemic injury and increased the number of BrdU-positive cells that differentiated into mature neurons in the ischemic striatum by day 28. Treatment with LV-Wnt3a-HA into the subventricular zone significantly enhanced functional recovery from the second day after injury and increased the number of immature neurons in the striatum and subventricular zone. This was accompanied by reduced dissemination of the neuronal injury. Our data indicate that Wnt signaling appears to contribute to functional recovery after ischemic injury by increasing neurogenesis or neuronal survival in the striatum.

Transplantation of placenta-derived mesenchymal stem cells in the EAE mouse model of MS.

Stem cell-based regenerative medicine raises great hope for the treatment of multiple sclerosis (MS). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are being tested in clinical trials. Bone marrow is the traditional source of human MSCs, but human term placenta appears to be an excellent alternative because of its availability, without ethical issues. In this study, the therapeutic effect of human placental MSCs (PL-MSCs) was evaluated in experimental autoimmune encephalomyelitis (EAE), the mice model of MS. EAE mice were transplanted intra-cerebrally with PL-MSCs or with the vehicle saline 5 or 10 days after first MOG injection. The mice were monitored for a month after therapy. A daily EAE score revealed a decrease in disease severity in the transplanted animals when compared to saline. Survival was significantly higher in the transplanted animals. In vitro experiments demonstrated that conditioned media from LPS-activated astrocytes stimulated PL-MSCs to express the gene TNF-α-stimulated gene/protein 6 (TSG-6). The same mRNA expression was obtained when PL-MSCs were exposed to TNF-α or IL1-ß. These results demonstrate that PL-MSCs have a therapeutic effect in the EAE mice model. We assume that this effect is caused by reduction of the anti-inflammatory protein, TSG-6, of the inflammatory damage.

Patient and caregiver perceptions of the social impact of advanced Parkinson's disease and dyskinesias.

Parkinson's disease (PD) exacts a physical and emotional toll on both patients and family. The aim of this study was to compare patient and caregiver perceptions of the social consequences of basic symptoms of PD and levodopa-induced dyskinesias. Forty patients with PD and dyskinesias and 35 of their caregivers completed a self-report questionnaire on the impact of PD and dyskinesias on their feelings of security and embarrassment and participation in family/social events, and indicated their preference for the "on" (with dyskinesias) or the "off" (without dyskinesias) state. The patients scored significantly higher than the caregivers did on the negative social impact of the disease in general (p = 0.002) and of the dyskinesias in particular (p = 0.03). Nevertheless, the patients expressed a significantly greater preference for the "on" state (83 %) than the caregivers (59 %) (p = 0.03). Preferences turned to be reverse in direction among spouse-caregivers who significantly preferred the "off" state (54 %) than the patients (25 %) (p = 0.04). Although patients have a worse perception of the effects of PD than their caregivers do, they prefer the more independent "on" state, whereas their caregivers prefer the "off" state.

Respiratory distress: an unrecognized non-motor phenomenon in patients with parkinsonism.

Although respiratory abnormalities are associated with parkinsonism, patients rarely complain of dyspnea. This study describes ten patients with parkinsonism and symptoms of dyspnea and respiratory distress that were unexplained by a pulmonary or cardiac abnormality or a psychological problem. Suggested underlying mechanisms are a central pathology affecting respiratory rhythm generation at the brainstem or lack of coordination of the respiratory muscles causing involuntary movements of the diaphragm. Dyspnea and respiratory distress should be included among the non-motor symptoms of parkinsonism.

Placental mesenchymal stromal cells induced into neurotrophic factor-producing cells protect neuronal cells from hypoxia and oxidative stress.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) may be useful in a range of clinical applications. The placenta has been suggested as an abundant, ethically acceptable, less immunogenic and easily accessible source of MSC. The aim of this study was to evaluate the capacity of induced placental MSC to differentiate into neurotrophic factor-producing cells (NTF) and their protective effect on neuronal cells. METHODS: MSC were isolated from placentas and characterized by fluorescence-activated cell sorting (FACS). The cells underwent an induction protocol to differentiate them into NTF. Analysis of the cellular differentiation was done using polymerase chain reactions (PCR), immunocytochemical staining and enzyme-linked immunosorbent assays (ELISA). Conditioned media from placental MSC (PL-MSC) and differentiated MSC (PL-DIFF) were collected and examined for their ability to protect neural cells. RESULTS: The immunocytochemical studies showed that the cells displayed typical MSC membrane markers. The cells differentiated into osteoblasts and adipocytes. PCR and immunohistology staining demonstrated that the induced cells expressed typical astrocytes markers and neurotrophic factors. Vascular endothelial growth factor (VEGF) levels were higher in the conditioned media from PL-DIFF compared with PL-MSC, as indicated by ELISA. Both PL-DIFF and PL-MSC conditioned media markedly protected neural cells from oxidative stress induced by H(2)O(2) and 6-hydroxydopamine. PL-DIFF conditioned medium had a superior effect on neuronal cell survival. Anti-VEGF antibodies (Bevacizumab) reduced the protective effect of the conditioned media from differentiated and undifferentiated MSC. CONCLUSIONS: This study has demonstrated a neuroprotective effect of MSC of placental origin subjected to an induction differentiation protocol. These data offer the prospect of using placenta as a source for stem cell-based therapies.