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This study evaluates the role of gamma interferon in in vitro B-cell activation. Two clones of an alloreactive helper T-cell line were equally effective in activating B cells polyclonally to proliferate and to mature to immunoglobulin secretion. One of these clones produced high levels of gamma interferon, while the other clone did not. From these data we conclude that gamma interferon plays no limiting role in T-dependent B-cell activation. [ABSTRACT FROM AUTHOR]

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., (© 2023. The Author(s).)

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.

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.

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., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2020 International Society for Neurochemistry.)

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., (Copyright © 2020 Consiglio nazionale delle ricerche CNR Italy. Published by Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: Conflict of interestThe authors declare that they have no conflicts of interest., (© The Author(s) 2020.)

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.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)