Does cognitive decline influence signing?

OBJECTIVE: The study explored the change in handwritten signature in neurodegenerative diseases by using of a rater-based approach. METHODS: Four independent observers were required to compare a pair of signatures (on average, 5 years elapsed between the two signatures) made by 103 patients (mean age 72 years) with Alzheimer's disease (AD) or frontotemporal dementia (FTD) and by 31 healthy participants (HC; mean age 73 years), judging their change according to a 0-1 rating scale (0 = similar or 1 = different). If a signature change was detected, the rater had also to report which signature features (spatial layout, omitted/added/switched letters or names, shape of letter, pen-flow) changed on the same 0-1 scale. For the AD and FTD groups, one signature was collected prior to the diagnosis of dementia, the other subsequent. RESULTS: A signature change was reported by raters in 36% of AD patients, 44% of FTD, and 17% of HC, with significant differences between both clinical groups and HC (vs. AD, p = .01; vs. FTD, p = .001). There was not a distinctive marker of the signature change (i.e., feature change) in patients with dementia. Moreover, the signature changes in neurological patients were unrelated to their clinical and demographic characteristics (age, sex, education, time elapsed between the two signatures, Mini-mental State Examination score). CONCLUSION: The findings suggest a resistance of handwritten signature in neurodegenerative diseases and in physiological aging, also suggesting that the signature may be an unreliable indicator of the cognitive status in AD and FTD, at least if subjectively evaluated.

Nucleoporin Nup358 Downregulation Tunes the Neuronal Excitability in Mouse Cortical Neurons.

Nucleoporins (NUPs) are proteins that comprise the nuclear pore complexes (NPCs). The NPC spans the nuclear envelope of a cell and provides a channel through which RNA and proteins move between the nucleus and the cytoplasm and vice versa. NUP and NPC disruptions have a great impact on the pathophysiology of neurodegenerative diseases (NDDs). Although the downregulation of Nup358 leads to a reduction in the scaffold protein ankyrin-G at the axon initial segment (AIS) of mature neurons, the function of Nup358 in the cytoplasm of neurons remains elusive. To investigate whether Nup358 plays any role in neuronal activity, we downregulated Nup358 in non-pathological mouse cortical neurons and measured their active and passive bioelectrical properties. We identified that Nup358 downregulation is able to produce significant modifications of cell-membrane excitability via voltage-gated sodium channel kinetics. Our findings suggest that Nup358 contributes to neuronal excitability through a functional stabilization of the electrical properties of the neuronal membrane. Hypotheses will be discussed regarding the alteration of this active regulation as putatively occurring in the pathophysiology of NDDs.

Negr1-Derived Peptides Trigger ALK Degradation and Halt Neuroblastoma Progression In Vitro and In Vivo.

Neuroblastoma is among the most common childhood cancers. Neuroblastoma in advanced stages is one of the most intractable pediatric cancers, notwithstanding the recent therapeutic advances. ALK mutations are among the leading cause of hereditary neuroblastoma and account for more than 14% of the somatically acquired alterations. ALK kinase activity is currently one of the main targets for pharmacological strategies. However, evidence from ALK fusion-positive lung cancer studies has shown that resistance to ALK inhibition arises during the therapy, causing a relapse within several years. IgLONs are membrane-bound proteins involved in cell-to-cell adhesion. The expression of the IgLON family results altered in different cancers. We found that the IgLON member Negr1 is downregulated in neuroblastoma. The ectopic overexpression of Negr1 impairs neuroblastoma growth in vitro and in vivo. Negr1 exists as a GPI-anchored membrane-bound protein and as a soluble protein released upon metalloprotease cleavage. We generated and characterized a panel of Negr1-derived peptides. The treatment with Negr1 protein and derived peptides induce ALK downregulation and halt neuroblastoma progression in vitro and in vivo.

Psychometrics, diagnostics and usability of Italian tools assessing behavioural and functional outcomes in neurological, geriatric and psychiatric disorders: a systematic review.

BACKGROUND: Psychometric instruments assessing behavioural and functional outcomes (BFIs) in neurological, geriatric and psychiatric populations are relevant towards diagnostics, prognosis and intervention. However, BFIs often happen not to meet methodological-statistical standards, thus lowering their level of recommendation in clinical practice and research. This work thus aimed at (1) providing an up-to-date compendium on psychometrics, diagnostics and usability of available Italian BFIs and (2) delivering evidence-based information on their level of recommendation. METHODS: This review was pre-registered (PROSPERO ID: CRD42021295430) and performed according to PRISMA guidelines. Several psychometric, diagnostic and usability measures were addressed as outcomes. Quality assessment was performed via an ad hoc checklist, the Behavioural and Functional Instrument Quality Assessment. RESULTS: Out of an initial N = 830 reports, 108 studies were included (N = 102 BFIs). Target constructs included behavioural/psychiatric symptoms, quality of life and physical functioning. BFIs were either self- or caregiver-/clinician-report. Studies in clinical conditions (including neurological, psychiatric and geriatric ones) were the most represented. Validity was investigated for 85 and reliability for 80 BFIs, respectively. Criterion and factorial validity testing were infrequent, whereas content and ecological validity and parallel forms were almost never addressed. Item response theory analyses were seldom carried out. Diagnostics and norms lacked for about one-third of BFIs. Information on administration time, ease of use and ceiling/floor effects were often unreported. DISCUSSION: Several available BFIs for the Italian population do not meet adequate statistical-methodological standards, this prompting a greater care from researchers involved in their development.

Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease.

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80% of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinson's disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knock-in mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular re-localization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

LRRK2 G2019S kinase activity triggers neurotoxic NSF aggregation.

Parkinson's disease is characterized by the progressive degeneration of dopaminergic neurons within the substantia nigra pars compacta and the presence of protein aggregates in surviving neurons. The LRRK2 G2019S mutation is one of the major determinants of familial Parkinson's disease cases and leads to late-onset Parkinson's disease with pleomorphic pathology, including α-synuclein accumulation and deposition of protein inclusions. We demonstrated that LRRK2 phosphorylates N-ethylmaleimide sensitive factor (NSF). We observed aggregates containing NSF in basal ganglia specimens from patients with Parkinson's disease carrying the G2019S variant, and in cellular and animal models expressing the LRRK2 G2019S variant. We found that LRRK2 G2019S kinase activity induces the accumulation of NSF in toxic aggregates. Of note, the induction of autophagy cleared NSF aggregation and rescued motor and cognitive impairment observed in aged hG2019S bacterial artificial chromosome (BAC) mice. We suggest that LRRK2 G2019S pathological phosphorylation impacts on NSF biochemical properties, thus causing the formation of cytotoxic protein inclusions.

LRRK2 at the pre-synaptic site: A 16-years perspective.

Parkinson's disease is a common neurodegenerative disorder and is clinically characterized by bradykinesia, rigidity, and resting tremor. Missense mutations in the leucine-rich repeat protein kinase-2 gene (LRRK2) are a recognized cause of inherited Parkinson's disease. The physiological and pathological impact of LRRK2 is still obscure, but accumulating evidence indicates that LRRK2 orchestrates diverse aspects of membrane trafficking, such as membrane fusion and vesicle formation and transport along actin and tubulin tracks. In the present review, we focus on the special relation between LRRK2 and synaptic vesicles. LRRK2 binds and phosphorylates key actors within the synaptic vesicle cycle. Accordingly, alterations in dopamine and glutamate transmission have been described upon LRRK2 manipulations. However, the different modeling strategies and phenotypes observed require a critical approach to decipher the outcome of LRRK2 at the pre-synaptic site.

A reliable strategy for single-cell RNA sequencing analysis using cryoconserved primary cortical cells.

BACKGROUND: The application of single-cell RNA sequencing (scRNASeq) represents a unique approach to identify hundreds to millions of cells in mammalian cortical multilayers at different stages of embryogenesis. ScRNASeq technology applied to neurological studies requires the use of fresh starting materials because standard cryopreservation methods do not guarantee high viability of cortical primary cells derived from dissected brain areas. NEW METHOD: Here we set up and validate an innovative strategy to perform scRNASeq studies in cryopreserved primary cortical cells isolated from E15.5 mouse embryo. In order to freeze cortical primary cells, we have employed Neurostore, a medium able to guarantee high viability and cell composition of embryonic cortex after thawing. COMPARISON WITH EXISTING METHODS: We showed for the first time the possibility to run scRNASeq experiments on primary cortical cells in an off-line set-up, ensuring cellular integrity and diversity. RESULTS: By trypan blue assay and flow cytometry analysis, we found that Neurostore-cryopreserved cortical cells showed approximately 95 % of viability. Satisfactory RNA recovery and cDNA libraries were achieved. Transcriptome sequencing of 35,763 cryoconserved single cells yielded a robust data-set, identifying 25 cell clusters in three biological samples. Prevalence of peculiar neural populations before and after the cryopreservation-resuscitation procedure was verified by marker gene expression and immunofluorescence analysis. CONCLUSIONS: Our findings support the evidence that frozen primary cortical cells can be successfully employed in scRNASeq experiments allowing an unprecedented flexibility in experimental procedures, such as sample preparation and subsequent processing steps performed in different locations.

Depression-Associated Gene Negr1-Fgfr2 Pathway Is Altered by Antidepressant Treatment.

The Negr1 gene has been significantly associated with major depression in genetic studies. Negr1 encodes for a cell adhesion molecule cleaved by the protease Adam10, thus activating Fgfr2 and promoting neuronal spine plasticity. We investigated whether antidepressants modulate the expression of genes belonging to Negr1-Fgfr2 pathway in Flinders sensitive line (FSL) rats, in a corticosterone-treated mouse model of depression, and in mouse primary neurons. Negr1 and Adam10 were the genes mostly affected by antidepressant treatment, and in opposite directions. Negr1 was down-regulated by escitalopram in the hypothalamus of FSL rats, by fluoxetine in the hippocampal dentate gyrus of corticosterone-treated mice, and by nortriptyline in hippocampal primary neurons. Adam10 mRNA was increased by nortriptyline administration in the hypothalamus, by escitalopram in the hippocampus of FSL rats, and by fluoxetine in mouse dorsal dentate gyrus. Similarly, nortriptyline increased Adam10 expression in hippocampal cultures. Fgfr2 expression was increased by nortriptyline in the hypothalamus of FSL rats and in hippocampal neurons. Lsamp, another IgLON family protein, increased in mouse dentate gyrus after fluoxetine treatment. These findings suggest that Negr1-Fgfr2 pathway plays a role in the modulation of synaptic plasticity induced by antidepressant treatment to promote therapeutic efficacy by rearranging connectivity in corticolimbic circuits impaired in depression.

Kinase inhibition of G2019S-LRRK2 enhances autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions.

The Parkinson's disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance.

The LRRK2 N-terminal domain influences vesicle trafficking: impact of the E193K variant.

The LRRK2 protein consists of multiple functional domains, including protein-binding domains at its N and C-terminus. Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) have been linked to familial and sporadic Parkinson's disease (PD). We have recently described a novel variant falling within the N-terminal armadillo repeats, E193K. Herein, our aim is to investigate the functional impact of LRRK2 N-terminal domain and the E193K variant on vesicle trafficking. By combining Total Internal Reflection Fluorescence (TIRF) microscopy and a synaptopHluorin assay, we found that expression of a construct lacking the N-terminal domain increases the frequency and amplitude of spontaneous synaptic events. Complementary biochemical approaches showed that the E193K variant alters the binding properties of LRRK2, decreases LRRK2 binding to synaptic vesicles, and promotes vesicle fusion. Our results confirm the physiological and pathological relevance of the nature of the LRRK2-associated macro-molecular complex solidifying the idea that different pathological mutations critically alter the scaffolding function of LRRK2 resulting in a perturbation of the vesicular trafficking as a common denominator.

Ankyrin-G induces nucleoporin Nup358 to associate with the axon initial segment of neurons.

Nup358 (also known as RanBP2) is a member of the large nucleoporin family that constitutes the nuclear pore complex. Depending on the cell type and the physiological state, Nup358 interacts with specific partner proteins and influences distinct mechanisms independent of its role in nucleocytoplasmic transport. Here, we provide evidence that Nup358 associates selectively with the axon initial segment (AIS) of mature neurons, mediated by the AIS scaffold protein ankyrin-G (AnkG, also known as Ank3). The N-terminus of Nup358 is demonstrated to be sufficient for its localization at the AIS. Further, we show that Nup358 is expressed as two isoforms, one full-length and another shorter form of Nup358. These isoforms differ in their subcellular distribution in neurons and expression level during neuronal development. Overall, the present study highlights an unprecedented localization of Nup358 within the AIS and suggests its involvement in neuronal function.This article has an associated First Person interview with the first author of the paper.

Leucine-rich repeat kinase 2 phosphorylation on synapsin I regulates glutamate release at pre-synaptic sites.

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain scaffolding protein with kinase and GTPase activities involved in synaptic vesicle (SV) dynamics. While its role in Parkinson's disease has been largely investigated, little is known about LRRK2 physiological role and until now few proteins have been described as substrates. We have previously demonstrated that LRRK2 through its WD40 domain interacts with synapsin I, an important SV-associated phosphoprotein involved in neuronal development and in the regulation of neurotransmitter release. To test whether synapsin I is substrate for LRRK2 and characterize the properties of its phosphorylation, we used in vitro kinase and binding assays as well as cellular model and site-direct mutagenesis. Using synaptosomes in superfusion, patch-clamp recordings in autaptic WT and synapsin I KO cortical neurons and SypHy assay on primary cortical culture from wild-type and BAC human LRRK2 G2019S mice we characterized the role of LRRK2 kinase activity on glutamate release and SV trafficking. Here we reported that synapsin I is phosphorylated by LRRK2 and demonstrated that the interaction between LRRK2 WD40 domain and synapsin I is crucial for this phosphorylation. Moreover, we showed that LRRK2 phosphorylation of synapsin I at threonine 337 and 339 significantly reduces synapsin I-SV/actin interactions. Using complementary experimental approaches, we demonstrated that LRRK2 controls glutamate release and SV dynamics in a kinase activity and synapsin I-dependent manner. Our findings show that synapsin I is a LRRK2 substrate and describe a novel mechanisms of regulation of glutamate release by LRRK2 kinase activity.

Parkin Interacts with Apoptosis-Inducing Factor and Interferes with Its Translocation to the Nucleus in Neuronal Cells.

Mutations in the PRKN gene (encoding parkin) have been linked to the most frequent known cause of recessive Parkinson's disease (PD), and parkin dysfunction represents a risk factor for sporadic PD. Parkin is widely neuroprotective through different cellular pathways, as it protects dopaminergic neurons from apoptosis in a series of cellular and animal models of PD. The mitochondrial protein apoptosis-inducing factor (AIF) is an important cell death effector, which, upon cellular stress in many paradigms, is redistributed from the mitochondria to the nucleus to function as a proapoptotic factor, mostly independent of caspase activity, while in normal mitochondria it functions as an antiapoptotic factor. AIF is known to participate in dopaminergic neuron loss in experimental PD models and in patients with PD. We, therefore, investigated possible crosstalk between parkin and AIF. By using immunoprecipitation and proximity ligation assays, we demonstrated a physical interaction between the two proteins. Nuclear AIF translocation was significantly reduced by parkin expression in neuroblastoma SH-SY5Y cells after exposure to an apoptogenic stimulus. These results were confirmed in primary murine cortical neurons, which showed a higher nuclear translocation of AIF in parkin-deficient neurons upon an excitotoxic stimulus. Our results indicate that the interaction of parkin with AIF interferes with the nuclear translocation of AIF, which might contribute to the neuroprotective activity of parkin.

Neurostore: A Novel Cryopreserving Medium for Primary Neurons.

Primary neuronal culture from rodents is a key tool in neurobiology. However, the preparation of primary cultures requires precise planning, starting from animal mating. Furthermore, each preparation generates a high amount of cells that eventually go wasted. The possibility to cryopreserve primary neural cells represents a resource for in vitro studies and significantly reduces the sacrifice of animals. Here we describe that Neurostore buffer supports the cryopreservation of primary neurons.

NEGR1 and FGFR2 cooperatively regulate cortical development and core behaviours related to autism disorders in mice.

Autism spectrum disorders are neurodevelopmental conditions with diverse aetiologies, all characterized by common core symptoms such as impaired social skills and communication, as well as repetitive behaviour. Cell adhesion molecules, receptor tyrosine kinases and associated downstream signalling have been strongly implicated in both neurodevelopment and autism spectrum disorders. We found that downregulation of the cell adhesion molecule NEGR1 or the receptor tyrosine kinase fibroblast growth factor receptor 2 (FGFR2) similarly affects neuronal migration and spine density during mouse cortical development in vivo and results in impaired core behaviours related to autism spectrum disorders. Mechanistically, NEGR1 physically interacts with FGFR2 and modulates FGFR2-dependent extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signalling by decreasing FGFR2 degradation from the plasma membrane. Accordingly, FGFR2 overexpression rescues all defects due to Negr1 knockdown in vivo. Negr1 knockout mice present phenotypes similar to Negr1-downregulated animals. These data indicate that NEGR1 and FGFR2 cooperatively regulate cortical development and suggest a role for defective NEGR1-FGFR2 complex and convergent downstream ERK and AKT signalling in autism spectrum disorders.

Preliminary results of the Multicenter Observational Study with Enoximone in Cardiac surgery (MOSEC).

BACKGROUND: Perioperative administration of Enoximone has been shown to improve hemodynamics, organ function, and inflammatory response. Aim of the present study was to evaluate the effects of Enoximone after on-pump cardiac surgery. METHODS: A protocol for a multicenter observational study was reviewed and approved by local ethic committee. This preliminary report involves the first 29 patients enrolled, in whom Enoximone was perioperatively administered in the context of on-pump cardiac surgery. All patients enrolled were propensity-matched 1:1 with controls not receiving Enoximone, renal function was evaluated in terms of estimated glomerular filtration rate (eGFR) with the CKD-EPI equation. RESULTS: After propensity matching, the two cohorts of patients receiving Enoximone or not did not show any significant differences among baseline characteristics. Patients receiving Enoximone showed a progressive improvement of eGFR at each time-point of follow-up: roughly +4.3, +10.0, and +12.3 mL/min/1.73 m2 on postoperative days 2, 7, and 30; respectively. Consistently, maximum difference versus baseline was +12.6 mL/min/1.73 m2 (or +19.3%) among Enoximone patients vs +3.3 mL/min/1.73 m2 (or +4.4%) among controls (p = 0.02). Multivariable regression analysis (R2-adjusted 0.47) showed only age (ß -0.53; p = 0.01), preoperative eGFR (ß -0.39; p = 0.02), diabetes (ß 2.1; p = 0.01), cardio-pulmonary bypass duration (ß 0.08; p = 0.05), and Enoximone administration (ß -0.74; p = 0.05) to be independently correlated with delta eGFR variation on day 30. CONCLUSION: These preliminary results show that perioperative Enoximone administration improved renal function in patients undergoing on-pump cardiac surgery. Further studies are needed to confirm these findings.

Cryopreservation of Primary Mouse Neurons: The Benefit of Neurostore Cryoprotective Medium.

Primary neuronal culture from rodents is a well-established model to investigate cellular neurobiology in vitro. However, for this purpose cell cultures need to be generated expressly, requiring extensive animal handling. Furthermore, often the preparation of fresh culture generates an excess of cells that are ultimately wasted. Therefore the ability to successfully cryopreserve primary neural cells would represent an important resource for neuroscience research and would allow to significantly reduce the sacrifice of animals. We describe here a novel freezing medium that allows long-term cryopreservation of primary mouse neurons prepared from E15.5 embryos. Combining imaging, biochemical and electrophysiological analyses, we found that cryopreserved cultures are viable and mature regarding morphology and functionality. These findings suggest that cryopreserved neurons are a valuable alternative to acutely dissociated neural cultures.

The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1.

Mutations in leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3ε. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.

The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.

Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson's disease (PD). LRRK2 protein contains several functional domains, including protein-protein interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely ß3-Cav2.1 at N-terminus part and ß-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause disease.