From cell lines to pluripotent Stem Cells for Modelling Parkinson's Disease

Parkinson's disease (PD) is the second most common neurodegenerative disorder characterized by loss of dopaminergic (DAergic) neurons in the substantia nigra that contributes to the main motor symptoms of the disease. At present, even if several advancements have been done in the last decades, the molecular and cellular mechanisms involved in the pathogenesis are far to be fully understood. Accordingly, the establishment of reliable in vitro experimental models to investigate the early events of the pathogenesis represents a key issue in the field. However, to mimic and reproduce in vitro the complex neuronal circuitry involved in PD-associated degeneration of DAergic neurons still remains a highly challenging issue. Here we will review the in vitro PD models used in the last 25 years of research, ranging from cell lines, primary rat or mice neuronal cultures to the more recent use of human induced pluripotent stem cells (hiPSCs) and, finally, the development of 3D midbrain organoids

Development of the Italian Version of the National Institutes of Health Stroke Scale It-NIHSS

Background and Purpose-The National Institutes of Health Stroke Scale (NIHSS) is a basic component of the assessment of patients with acute stroke. To foster and standardize the use of the NIHSS among Italian health professionals, we translated the scale, dubbed into Italian the training and test videotapes devised by the National Institutes of Health researchers, and conducted a series of certification courses using the translated videos. Methods-Translation, text adaptation, video dubbing, and editing of the Italian NIHSS videotapes relied on a team of bilingual stroke neurologists. Three waves of training courses were organized for mixed classes of medical and nonmedical health professionals. The certification test was based on the usual set of 5 videotaped patients. Scoring rules were those provided by the National Institutes of Neurological Disorders and Stroke. Reliability of the Italian NIHSS was assessed using kappa statistics and compared with that of the original NIHSS. Results-During 3 years, 850 nurses, 460 nonneurologist physicians, and 246 neurologists were trained. Pass rates were respectively 44%, 75%, and 87%, respectively. Overall, 80% of scale items showed moderate to excellent reliability. Independent significant predictors of test failure at multivariate logistic regression were nurse profession (OR, 5.41; 95% CI, 4.07 to 7.20), older age (OR, 1.03; 95% CI, 1.02 to 1.05), and first edition of the course (OR, 3.13; 95% CI, 2.43 to 4.05). The agreement across all items between NIHSS and the Italian NIHSS was 80% (kappa = 0.70 +/- 0.18, z < 0.001). Conclusions-The Italian translation, supervised by experienced vascular neurologists, did not influence the clinimetric characteristics of the NIHSS. Our findings support the implementation of NIHSS video training in languages other than English. (Stroke. 2009; 40: 2557-2559.

The evaluation of tactile dysfunction in the hand in type 1 diabetes: a novel method based on haptics

Aims We present an innovative method based on haptics for the evaluation of the sense of touch in the hand, in people affected by type 1 diabetes. Methods Forty individuals affected by diabetes and 20 healthy controls took part in the study; the diabetes group was further divided into two subgroups based on vibration sensitivity in the lower limb. By means of a novel haptic device, tactile sensitivity in the fingertip was measured as the ability of the participants to discriminate slip motion speed. Results Tactile sensitivity was significantly lower in individuals affected by diabetes as compared to controls. Depending on the subgroup, the difference from the controls was equal to 0.11 (95% CI from 0.029 to 0.186) and to 0.267 (95% CI from 0.198 to 0.336). Within the diabetes group, tactile sensitivity correlated with vibration sensitivity in the upper (p = 0.001) and lower limb (p = 0.003). A significant relationship between nerve conduction parameters and tactile sensitivity was found (p = 0.03). Finally, we combined the different predictors (clinical, vibratory and electroneurography data) by using cluster analysis; tactile sensitivity was found to be significantly different between different clusters (p = 0.004). Conclusions Early signs of tactile dysfunction in the hand were found in individuals affected by diabetes, even in absence of diabetic neuropathy. The protocol presented in this study is a promising tool for the assessment of tactile dysfunction in the hand in people affected by type 1 diabetes

Phosphoproteomic Landscaping Identifies Non-canonical cKIT Signaling in Polycythemia Vera Erythroid Progenitors

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV

Serotonin drives striatal synaptic plasticity in a sex-related manner

Introduction: Plasticity at corticostriatal synapses is a key substrate for a variety of brain functions – including motor control, learning and reward processing – and is often disrupted in disease conditions. Despite intense research pointing toward a dynamic interplay between glutamate, dopamine (DA), and serotonin (5-HT) neurotransmission, their precise circuit and synaptic mechanisms regulating their role in striatal plasticity are still unclear. Here, we analyze the role of serotonergic raphe-striatal innervation in the regulation of DA-dependent corticostriatal plasticity. Methods: Mice (males and females, 2–6 months of age) were housed in standard plexiglass cages at constant temperature (22 ± 1 °C) and maintained on a 12/12 h light/dark cycle with food and demineralized water ad libitum. In the present study, we used a knock-in mouse line in which the green fluorescent protein reporter gene (GFP) replaced the I Tph2 exon (Tph2GFP mice), allowing selective expression of GFP in the whole 5-HT system, highlighting both somata and neuritis of serotonergic neurons. Heterozygous, Tph2+/GFP, mice were intercrossed to obtain experimental cohorts, which included Wild-type (Tph2+/+), Heterozygous (Tph2+/GFP), and Mutant serotonin-depleted (Tph2GFP/GFP) animals. Results: Using male and female mice, carrying on different Tph2 gene dosages, we show that Tph2 gene modulation results in sex-specific corticostriatal abnormalities, encompassing the abnormal amplitude of spontaneous glutamatergic transmission and the loss of Long Term Potentiation (LTP) in Tph2GFP/GFP mice of both sexes, while this form of plasticity is normally expressed in control mice (Tph2+/+). Once LTP is induced, only the Tph2+/GFP female mice present a loss of synaptic depotentiation. Conclusion: We showed a relevant role of the interaction between dopaminergic and serotonergic systems in controlling striatal synaptic plasticity. Overall, our data unveil that 5-HT plays a primary role in regulating DA-dependent corticostriatal plasticity in a sex-related manner and propose altered 5-HT levels as a critical determinant of disease-associated plasticity defects

Derangement of Ras-Guanine Nucleotide-Releasing Factor 1 (Ras-GRF1) and Extracellular Signal-Regulated Kinase (ERK) Dependent Striatal Plasticity in L-DOPA-Induced Dyskinesia

BACKGROUND: Bidirectional long-term plasticity at the corticostriatal synapse has been proposed as a central cellular mechanism governing dopamine-mediated behavioral adaptations in the basal ganglia system. Balanced activity of medium spiny neurons (MSNs) in the direct and the indirect pathways is essential for normal striatal function. This balance is disrupted in Parkinson's disease and in L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID), a common motor complication of current pharmacotherapy of Parkinson's disease. METHODS: Electrophysiological recordings were performed in mouse cortico-striatal slice preparation. Synaptic plasticity, such as long-term potentiation (LTP) and depotentiation, was investigated. Specific pharmacological inhibitors or genetic manipulations were used to modulate the Ras-extracellular signal-regulated kinase (Ras-ERK) pathway, a signal transduction cascade implicated in behavioral plasticity, and synaptic activity in different subpopulations of striatal neurons was measured. RESULTS: We found that the Ras-ERK pathway, is not only essential for long-term potentiation induced with a high frequency stimulation protocol (HFS-LTP) in the dorsal striatum, but also for its reversal, synaptic depotentiation. Ablation of Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a neuronal activator of Ras proteins, causes a specific loss of HFS-LTP in the medium spiny neurons in the direct pathway without affecting LTP in the indirect pathway. Analysis of LTP in animals with unilateral 6-hydroxydopamine lesions (6-OHDA) rendered dyskinetic with chronic L-DOPA treatment reveals a complex, Ras-GRF1 and pathway-independent, apparently stochastic involvement of ERK. CONCLUSIONS: These data not only demonstrate a central role for Ras-ERK signaling in striatal LTP, depotentiation, and LTP restored after L-DOPA treatment but also disclose multifaceted synaptic adaptations occurring in response to dopaminergic denervation and pulsatile administration of L-DOPA

Phosphoproteomic Landscaping Identifies Non-canonical cKIT Signaling in Polycythemia Vera Erythroid Progenitors

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV

Generation and quality control of lipidomics data for the alzheimers disease neuroimaging initiative cohort.

Alzheimers disease (AD) is a major public health priority with a large socioeconomic burden and complex etiology. The Alzheimer Disease Metabolomics Consortium (ADMC) and the Alzheimer Disease Neuroimaging Initiative (ADNI) aim to gain new biological insights in the disease etiology. We report here an untargeted lipidomics of serum specimens of 806 subjects within the ADNI1 cohort (188 AD, 392 mild cognitive impairment and 226 cognitively normal subjects) along with 83 quality control samples. Lipids were detected and measured using an ultra-high-performance liquid chromatography quadruple/time-of-flight mass spectrometry (UHPLC-QTOF MS) instrument operated in both negative and positive electrospray ionization modes. The dataset includes a total 513 unique lipid species out of which 341 are known lipids. For over 95% of the detected lipids, a relative standard deviation of better than 20% was achieved in the quality control samples, indicating high technical reproducibility. Association modeling of this dataset and available clinical, metabolomics and drug-use data will provide novel insights into the AD etiology. These datasets are available at the ADNI repository at http://adni.loni.usc.edu/

Impact of Glycemic and Blood Pressure Variability on Surrogate Measures of Cardiovascular Outcomes in Type 2 Diabetic Patients

OBJECTIVE—The effect of glycemic variability (GV) on cardiovascular risk has not been fully clarified in type 2 diabetes. We evaluated the effect of GV, blood pressure (BP), and oxidative stress on intima-media thickness (IMT), left ventricular mass index (LVMI), flow-mediated dilation (FMD), and sympathovagal balance (low frequency [LF]/high frequency [HF] ratio) in 26 type 2 diabetic patients (diabetes duration 4.41 6 4.81 years; HbA1c 6.70 6 1.25%) receiving diet and/or metformin treatment, with no hypotensive treatment or complications. RESEARCH DESIGN AND METHODS—Continuous glucose monitoring (CGM) data were used to calculate mean amplitude of glycemic excursion (MAGE), continuous overall net glycemic action (CONGA)-2, mean blood glucose (MBG), mean postprandial glucose excursion (MPPGE), and incremental area under the curve (IAUC). Blood pressure (BP), circadian rhythm, and urinary 15-F2t-isoprostane (8-iso-prostaglandin F2a [PGF2a]) were also evaluated. Subjects were divided into dipper (D) and nondipper (ND) groups according to DBP. RESULTS—IMT and LVMIwere increased inNDversusD(0.7760.08 vs. 0.6860.13 [P=0.04] and 67 6 14 vs. 55 6 11 [P = 0.03], respectively). MBG, MAGE, and IAUC were significantly associated with LF/HF ratio at night (r = 0.50, P = 0.01; r = 0.40, P = 0.04; r = 0.41, P = 0.04, respectively), MPPGE was negatively associated with FMD (r =20.45, P = 0.02), andCONGA-2was positively associatedwith LVMI (r=0.55, P=0.006).TheDsystolic BP was negatively associated with IMT (r =20.43, P = 0.03) andwith LVMI (r =20.52, P = 0.01). Urinary 8-iso-PGF2a was positively associated with LVMI (r = 0.68 P , 0.001). CONCLUSIONS—An impaired GV and BP variability is associated with endothelial and cardiovascular damage in short-term diabetic patients with optimal metabolic control. Oxidative stress is the only independent predictor of increased LV mass and correlates with glucose and BP variability

Rabphilin 3A: A novel target for the treatment of levodopa-induced dyskinesias

N-methyl-d-aspartate receptor (NMDAR) subunit composition strictly commands receptor function and pharmacological responses. Changes in NMDAR subunit composition have been documented in brain disorders such as Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesias (LIDs), where an increase of NMDAR GluN2A/GluN2B subunit ratio at striatal synapses has been observed. A therapeutic approach aimed at rebalancing NMDAR synaptic composition represents a valuable strategy for PD and LIDs. To this, the comprehension of the molecular mechanisms regulating the synaptic localization of different NMDAR subtypes is required. We have recently demonstrated that Rabphilin 3A (Rph3A) is a new binding partner of NMDARs containing the GluN2A subunit and that it plays a crucial function in the synaptic stabilization of these receptors. Considering that protein-protein interactions govern the synaptic retention of NMDARs, the purpose of this work was to analyse the role of Rph3A and Rph3A/NMDAR complex in PD and LIDs, and to modulate Rph3A/GluN2A interaction to counteract the aberrant motor behaviour associated to chronic L-DOPA administration. Thus, an array of biochemical, immunohistochemical and pharmacological tools together with electron microscopy were applied in this study. Here we found that Rph3A is localized at the striatal postsynaptic density where it interacts with GluN2A. Notably, Rph3A expression at the synapse and its interaction with GluN2A-containing NMDARs were increased in parkinsonian rats displaying a dyskinetic profile. Acute treatment of dyskinetic animals with a cell-permeable peptide able to interfere with Rph3A/GluN2A binding significantly reduced their abnormal motor behaviour. Altogether, our findings indicate that Rph3A activity is linked to the aberrant synaptic localization of GluN2A-expressing NMDARs characterizing LIDs. Thus, we suggest that Rph3A/GluN2A complex could represent an innovative therapeutic target for those pathological conditions where NMDAR composition is significantly altered