VPS35 mutations in Parkinson disease.

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD.

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.

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.

A double-blind, delayed-start trial of rasagiline in Parkinson's disease.

BACKGROUND: A therapy that slows disease progression is the major unmet need in Parkinson's disease. METHODS: In this double-blind trial, we examined the possibility that rasagiline has disease-modifying effects in Parkinson's disease. A total of 1176 subjects with untreated Parkinson's disease were randomly assigned to receive rasagiline (at a dose of either 1 mg or 2 mg per day) for 72 weeks (the early-start group) or placebo for 36 weeks followed by rasagiline (at a dose of either 1 mg or 2 mg per day) for 36 weeks (the delayed-start group). To determine a positive result with either dose, the early-start treatment group had to meet each of three hierarchical end points of the primary analysis based on the Unified Parkinson's Disease Rating Scale (UPDRS, a 176-point scale, with higher numbers indicating more severe disease): superiority to placebo in the rate of change in the UPDRS score between weeks 12 and 36, superiority to delayed-start treatment in the change in the score between baseline and week 72, and noninferiority to delayed-start treatment in the rate of change in the score between weeks 48 and 72. RESULTS: Early-start treatment with rasagiline at a dose of 1 mg per day met all end points in the primary analysis: a smaller mean (+/-SE) increase (rate of worsening) in the UPDRS score between weeks 12 and 36 (0.09+/-0.02 points per week in the early-start group vs. 0.14+/-0.01 points per week in the placebo group, P=0.01), less worsening in the score between baseline and week 72 (2.82+/-0.53 points in the early-start group vs. 4.52+/-0.56 points in the delayed-start group, P=0.02), and noninferiority between the two groups with respect to the rate of change in the UPDRS score between weeks 48 and 72 (0.085+/-0.02 points per week in the early-start group vs. 0.085+/-0.02 points per week in the delayed-start group, P<0.001). All three end points were not met with rasagiline at a dose of 2 mg per day, since the change in the UPDRS score between baseline and week 72 was not significantly different in the two groups (3.47+/-0.50 points in the early-start group and 3.11+/-0.50 points in the delayed-start group, P=0.60). CONCLUSIONS: Early treatment with rasagiline at a dose of 1 mg per day provided benefits that were consistent with a possible disease-modifying effect, but early treatment with rasagiline at a dose of 2 mg per day did not. Because the two doses were associated with different outcomes, the study results must be interpreted with caution. (ClinicalTrials.gov number, NCT00256204.)

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.

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.

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.

Wnt signaling pathway overcomes the disruption of neuronal differentiation of neural progenitor cells induced by oligomeric amyloid ß-peptide.

Neural stem cells give rise to new hippocampal neurons throughout adulthood. Defects in neurogenesis are associated with cognitive dysfunctions, such as Alzheimer disease (AD). Our understanding of the signals controlling this process is limited. The present in vitro study explored the manner in which the Wnt signaling pathway regulates the differentiation of hippocampal progenitors (HPs) into neurons under the influence of amyloid ß(42) (Aß(42) ). The results showed that oligomeric Aß(42) reduced neuronal differentiation. This process was accompanied by a reduction in active ß-catenin levels and proneural gene expression. The addition of Wnt3a increased the neuronal differentiation of Aß(42) -treated HPs, at the expense of astrocyte differentiation. The effect of Wnt signaling was attributable to progenitor cell differentiation to the neuronal lineage, and not to increased proliferation or rescue of neurons. The interruption of Wnt signaling by oligomeric Aß(42) may have clinical implications for the treatment of impaired neurogenesis in AD.

Residual striatal dopaminergic nerve terminals in very long-standing Parkinson's disease: a single photon emission computed tomography imaging study.

Molecular imaging studies of Parkinson's disease (PD) progression mostly focus on the first 5 years after disease onset, demonstrating rapid initial nigrostriatal neuronal loss. The fate of residual functional dopaminergic nerve terminals in patients with long-standing PD has not yet been specifically explored. Therefore, we performed [(123)I]-FP-CIT single photon emission computed tomography (SPECT) in 15 patients with very long-standing PD (mean disease duration 20.6 ± 6.3 years). Measurable uptake of [(123)I]-FP-CIT was still detected in the striata of all patients. As seen in early stages, reduction of tracer uptake in the putamen was more prominent than in the caudate nucleus. Asymmetry in tracer uptake between the two putamen and caudate nuclei was preserved. These findings indicate that degeneration of dopaminergic neurons in PD is not total even after many years of illness. Data should be considered in exploring underlying causes of progressive loss of nigrostriatal dopaminergic neurons and development of future novel dopaminergic therapeutic strategies in PD.

Neurogenesis in the aged and neurodegenerative brain.

It has been well established that adult neurogenesis occurs throughout life in the subventricular (SVZ) and subgranular (SGZ) zones. However, the exact role of this type of brain plasticity is not yet clear. Many studies have shown that neurogenesis is involved in learning and memory. This has led to a hypothesis which suggests that impairment in memory during aging and neurodegenerative diseases such as Alzheimer's disease (AD) may involve abnormal neurogenesis. Indeed, during aging, there is an age-related decline in adult neurogenesis. This decline is mostly related to decreased proliferation, associated to decreased stimulation to proliferate in an aging brain. In AD, there is also evidence for decreased neurogenesis, that accompanies the neuronal loss characteristic of the disease. Interestingly in AD, there is increased proliferation, that may be caused by increasing amounts of soluble amyloid ß42-protein (Aß42). However, most of these new neurons die, and fibrillar Aß42 seems to be involved in generating an inappropriate environment for these neurons to mature. These findings open prospects for new strategies that can increase neurogenesis in normal or pathological processes in the aging brain, and by that decrease memory deficits.

Longitudinal MRI and MRSI characterization of the quinolinic acid rat model for excitotoxicity: peculiar apparent diffusion coefficients and recovery of N-acetyl aspartate levels.

Quinolinic acid (QA) induced striatal lesion is an important model for excitotoxicity that is also used for efficacy studies. To date, the morphological and spectroscopic indices of this model have not been studied longitudinally by MRI; therefore the objectives of this study were aimed at following the lesion progression and changes in N-acetyl aspartate (NAA) as viewed by MRI and MRSI, respectively, in-vivo over a period of 49 days. We found that the affected areas exhibited both high and low apparent diffusion coefficients (ADC) even 49 days post QA injection in three of the six tested animals. MRI-guided histological analysis correlated areas characterized by high ADCs on day 49 with cellular loss, while areas characterized by lower ADCs were correlated with macrophage infiltration (CD68 positive stain). Our MRSI study revealed an initial reduction of NAA levels in the lesioned striatum, which significantly recovered with time, although not to control levels. Total-striatum normalized NAA levels recovered from 0.67 +/- 0.15 (of the contralateral row) on day 1 to 0.90 +/- 0.12 on day 49. Our findings suggest that NAA should be considered as a marker for neuronal dysfunction, in addition to neuronal viability. Some behavioral indices could be correlated to permanent neuronal damage while others demonstrated a spontaneous recovery parallel to the NAA recovery. Our findings may have implications in efficacy-oriented studies performed on the QA model.

Introducing transcription factors to multipotent mesenchymal stem cells: making transdifferentiation possible.

Multipotent mesenchymal stem cells (MSCs) represent a promising autologous source for regenerative medicine. Because MSCs can be isolated from adult tissues, they represent an attractive cell source for autologous transplantation. A straightforward therapeutic strategy in the field of stem cell-based regenerative medicine is the transplantation of functional differentiated cells as cell replacement for the lost or defective cells affected by disease. However, this strategy requires the capacity to regulate stem cell differentiation toward the desired cell fate. This therapeutic approach assumes the capability to direct MSC differentiation toward diverse cell fates, including those outside the mesenchymal lineage, a process termed transdifferentiation. The capacity of MSCs to undergo functional transdifferentiation has been questioned over the years. Nonetheless, recent studies support that genetic manipulation can serve to promote transdifferentiation. Specifically, forced expression of certain transcription factors can lead to reprogramming and alter cell fate. Using such a method, fully differentiated lymphocytes have been reprogrammed to become macrophages and, remarkably, somatic cells have been reprogrammed to become embryonic stem-like cells. In this review, we discuss the past and current research aimed at transdifferentiating MSCs, a process with applications that could revolutionize regenerative medicine.

Unilateral lower limb rest tremor is not necessarily a presenting symptom of Parkinson's disease.

Lower leg rest tremor is an uncommon symptom of neurological disease. Review of the files of 16 patients who presented with lower leg tremor (average age 58 +/- 16 years; average disease duration 6.8 +/- 8.5 years) yielded a diagnosis of Parkinson's disease (PD) in 5 and probable multiple system atrophy (MSA) in 3. In 4 patients with an indeterminate diagnosis, cardiac MIBG SPECT was positive in 3, indicating PD, and negative in one, suggesting MSA. Two patients each had psychogenic tremor and drug-induced parkinsonism. Although lower leg tremor is considered an unusual presentation of PD, it should raise suspicions of MSA and other neurodegenerative disorders.

DJ-1 changes in G93A-SOD1 transgenic mice: implications for oxidative stress in ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder. The causes of ALS are still obscure. Accumulating evidence supports the hypothesis that oxidative stress and mitochondrial dysfunction can be implicated in ALS pathogenesis. DJ-1 plays an important role in the oxidative stress response. The aim of this study was to discover whether there are changes in DJ-1 expression or in DJ-1-oxidized isoforms in an animal model of ALS. We used mutant SOD1(G93A) transgenic mice, a commonly used animal model for ALS. Upregulation of DJ-1 mRNA and protein levels were identified in the brains and spinal cords of SOD1(G93A) transgenic mice as compared to wild-type controls, evident from an early disease stage. Furthermore, an increase in DJ-1 acidic isoforms was detected, implying that there are more oxidized forms of DJ-1 in the CNS of SOD1(G93A) mice. This is the first report of possible involvement of DJ-1 in ALS. Since DJ-1 has a protective role against oxidative stress, it may suggest a possible therapeutic target in ALS.

Spinal cord mRNA profile in patients with ALS: comparison with transgenic mice expressing the human SOD-1 mutant.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons in the cerebral cortex, brain stem, and spinal cord. Most cases (90%) are classified as sporadic ALS (sALS). The remainder 10% are inherited and referred to as familial ALS, and 2% of instances are due to mutations in Cu/Zn superoxide dismutase (SOD1). Using cDNA microarray on postmortem spinal cord specimens of four sALS patients compared to four age-matched nonneurological controls, we found major changes in the expression of mRNA in 60 genes including increase of cathepsin B and cathepsin D (by the factors 2 and 2.3, respectively), apolipoprotein E (Apo E; factor 4.2), epidermal growth factor receptor (factor 10), ferritin (factor 2), and lysosomal trafficking regulator (factor 10). The increase in the expression of these genes was verified by quantitative reverse transcriptase polymerase chain reaction. Further analysis of these genes in hSOD1-G93A transgenic mice revealed increase in the expression in parallel with the deterioration of motor functions quantified by means of rotorod performance. The comparability of the findings in sALS patients and in the hSOD1-G93A transgenic mouse model suggests that the examined genes may play a specific role in the pathogenesis of ALS.

Migration of neurotrophic factors-secreting mesenchymal stem cells toward a quinolinic acid lesion as viewed by magnetic resonance imaging.

Stem cell-based treatment is a promising frontier for neurodegenerative diseases. We propose a novel protocol for inducing the differentiation of rat mesenchymal stem cells (MSCs) toward neurotrophic factor (NTF)-secreting cells as a possible neuroprotective agent. One of the major caveats of stem cell transplantation is their fate post-transplantation. To test the viability of the cells, we tracked the transplanted cells in vivo by magnetic resonance imaging (MRI) scans and validated the results by histology. MSCs went through a two-step medium-based differentiation protocol, followed by in vitro characterization using immunocytochemistry and immunoblotting analysis of the cell media. We examined the migratory properties of the cells in the quinolinic acid (QA)-induced striatal lesion model for Huntington's disease. The induced cells were labeled and transplanted posterior to the lesion. Rats underwent serial MRI scans to detect cell migration in vivo. On the 19th day, animals were sacrificed, and their brains were removed for immunostaining. Rat MSCs postinduction exhibited both neuronal and astrocyte markers, as well as production and secretion of NTFs. High-resolution two-dimensional and three-dimensional magnetic resonance images revealed that the cells migrated along a distinct route toward the lesion. The in vivo MRI results were validated by the histological study, which demonstrated that phagocytosis had only partially occurred and that MRI could correctly depict the status of the migrating cells. The results show that these cells migrated toward a QA lesion and therefore survived for 19 days post-transplantation. This gives hope for future research harnessing these cells for treating neurodegenerative diseases. Disclosure of potential conflicts of interest is found at the end of this article.

Comparative characterization of bone marrow-derived mesenchymal stromal cells from four different rat strains.

BACKGROUND AIMS: Bone marrow (BM) multipotent mesenchymal stromal cells (MSC) hold great potential for cell-based regenerative medicine. Because of the growing use of autologous rat MSC transplantation in various rat models, there is a need to establish minimal criteria for rat MSC characterization independent of the specific strain employed in each study. We aimed to compare the phenotypic and functional traits of BM MSC from the four strains of rats commonly used in research: Fisher, Lewis, Sprague-Dawley and Wistar. METHODS: Rat MSC were isolated from the BM of the four different rat strains in an identical fashion. Cells were characterized for their cell-surface phenotype in early and late passage. Functional mesenchymal differentiation capacities were examined following adipogenic and osteogenic inductions. Population doubling times were determined across the four strains throughout 10 passages. In vitro proliferation assays of immune cells were conducted following co-culture of spleen cells and MSC of the four different strains. RESULTS: We found that rat MSC from different strains exhibited similar cell-surface phenotype. Expansion rates and differentiation capacities of the MSC were also similar across the different strains. Co-culture of rat MSC with spleen cells obtained from rats of a different strain did not induce proliferation of immune cells. CONCLUSIONS: Our findings suggest that BM-derived MSC from different strains share similar characteristics, in contrast to the variations previously described in the characterization of mice MSC from different strains.

Lesions outside the CNS in Parkinson's disease.

Parkinson's disease (PD) is not a simple movement disorder induced just by loss of dopaminergic neurons in the substantia nigra pars compacta. Apparently, the substantia nigra is not the only or the first brain region damaged in PD. Moreover, older and recent studies have shown that the degenerative process in PD is much more extensive and affects not only the central nervous system (CNS) but also the peripheral autonomic nervous system and the organs outside the brain that the latter innervates. These include mainly the gastrointestinal tract, the heart, kidneys, urogenital system, and skin. Additional extra-CNS organs that are involved in PD include the eye and the adrenal gland. This article reviews the anatomical, physiological, and clinical features of extracerebral manifestations of PD, and describes their relevance to the etiology and pathogenesis of the disease. It establishes this illness as a systemic CNS and peripheral disorder that warrants new hypotheses regarding its causation and progression.

Validation of the freezing of gait questionnaire in patients with Parkinson's disease.

To revalidate the Freezing of Gait Questionnaire (FOG-Q), patients with Parkinson's disease (PD) were randomly assigned to receive rasagiline (1 mg/day) (n = 150), entacapone (200 mg with each dose of levodopa) (n = 150), or placebo (n = 154). Patients were assessed at baseline and after 10 weeks using the FOG-Q, Unified Parkinson's Disease Rating Scale (UPDRS), Beck Depression Inventory (BDI), and Parkinson's Disease Questionnaire (PDQ-39). FOG-Q dimensionality, test-retest reliability, and internal reliability were examined. Convergent and divergent validities were assessed by correlating FOG-Q with UPDRS, BDI, and PDQ-39. Comparisons between FOG-Q item 3 and UPDRS item 14 were also made. Principal component analysis indicated that FOG-Q measures a single dimension. Test-retest reliability and internal reliability of FOG-Q score was high. FOG-Q was best correlated to items of the UPDRS relating to walking, general motor issues, and mobility. Correlations between baseline and endpoint suggested that FOG-Q item 3 is at least as reliable as UPDRS item 14. At baseline, 85.9% of patients were identified as "Freezers" using FOG-Q item 3 (> or =1) and 44.1% using UPDRS item 14 (> or =1) (P < 0.001). FOG-Q was a reliable tool for the assessment of treatment intervention. FOG-Q item 3 was effective as a screening question for the presence of FOG.