Publications by authors named "Giovanni Piccoli"

56 Publications

Hypoglycemia due to PI3K/AKT/mTOR signaling pathway defects: two novel cases and review of the literature.

Hormones (Athens) 2021 Apr 20. Epub 2021 Apr 20.

Division of Pediatrics, S. Chiara General Hospital, Largo Medaglie d'oro, 9, 38122, Trento, Italy.

Introduction: The PI3K/AKT/mTOR signaling pathway is important for the regulation of multiple biological processes, including cellular growth and glucose metabolism. Defects of the PI3K/AKT/mTOR signaling pathway are not usually considered among the genetic causes of recurrent hypoglycemia in childhood. However, accumulating evidence links hypoglycemia with defects of this pathway.

Case Reports And Review: We describe here two cases of macrocephaly and hypoglycemia bearing genetic defects in genes involved in the PI3K/AKT/mTOR pathway. The first patient was diagnosed with a PTEN hamartoma tumour syndrome (PTHS) due to the de novo germline missense mutation c.[492 + 1G > A] of the PTEN gene. The second patient presented the autosomal dominant mental retardation-35 (MDR35) due to the heterozygous missense mutation c.592G > A in the PPP2R5D gene. A review of the literature on hypoglycemia and PI3K/AKT/mTOR signaling pathway defects, with a special focus on the metabolic characterization of hypoglycemia, is included.

Conclusions: PI3K/AKT/mTOR pathway defects should be included in the differential diagnosis of patients with hypoglycemia and macrocephaly. Clinical suspicion and molecular confirmation are important, not just for an accurate genetic counselling but also for defining the follow-up management, including cancer surveillance. The biochemical profile of hypoglycemia varies among patients. While most patients are characterized by low plasmatic insulin levels, hyperinsulinemia has also been observed. Large patient cohorts are needed to gain a comprehensive profile of the biochemical patterns of hypoglycemia in such defects and eventually guide targeted therapeutic interventions.
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http://dx.doi.org/10.1007/s42000-021-00287-1DOI Listing
April 2021

LRRK2 G2019S kinase activity triggers neurotoxic NSF aggregation.

Brain 2021 Apr 20. Epub 2021 Apr 20.

CIBIO, Università degli Studi di Trento, Trento, Italy.

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.
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http://dx.doi.org/10.1093/brain/awab073DOI Listing
April 2021

Trehalose-based neuroprotective autophagy inducers.

Bioorg Med Chem Lett 2021 May 9;40:127929. Epub 2021 Mar 9.

Istituto di Scienze e Tecnologie Chimiche (SCITEC) "Giulio Natta", Consiglio Nazionale delle Ricerche (CNR), Via C. Golgi 19, I-20133 Milan, Italy and Via G. Fantoli 16/15, I-20138 Milan, Italy. Electronic address:

A small set of trehalose-centered putative autophagy inducers was rationally designed and synthesized, with the aim to identify more potent and bioavailable autophagy inducers than free trehalose, and to acquire information about their molecular mechanism of action. Several robust, high yield routes to key trehalose intermediates and small molecule prodrugs (2-5), putative probes (6-10) and inorganic nanovectors (12a - thiol-PEG-triazole-trehalose constructs 11) were successfully executed, and compounds were tested for their autophagy-inducing properties. While small molecules 2-11 showed no pro-autophagic behavior at sub-millimolar concentrations, trehalose-bearing PEG-AuNPs 12a caused measurable autophagy induction at an estimated 40 μM trehalose concentration without any significant toxicity at the same concentration.
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http://dx.doi.org/10.1016/j.bmcl.2021.127929DOI Listing
May 2021

Gut Microbiota Status in COVID-19: An Unrecognized Player?

Front Cell Infect Microbiol 2020 26;10:576551. Epub 2020 Nov 26.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, Italy.

Infection with the SARS-CoV-2 virus causes cardiopulmonary and vascular complications, ranging in severity. Understanding the pathogenic mechanisms of the novel SARS-CoV2 infection and progression can provide potential novel targets for its prevention and/or treatment. Virus microbiota reciprocal interactions have been studied in a variety of viral infections. For example, the integrity of Coronavirus particles can be disrupted by surfactin, a bacterial surface molecule that targets other viruses, including that of influenza A. In this light, intestinal microbiota likely influences COVID-19 virulence, while from its side SARS-CoV-2 may affect the intestinal microbiome promoting dysbiosis and other deleterious consequences. Hence, the microbiota pre-existing health status and its alterations in the course of SARS-CoV-2 infection, are likely to play an important, still underscored role in determining individual susceptibility and resilience to COVID-19. Indeed, the vast majority of COVID-19 worst clinical conditions and fatalities develop in subjects with specific risk factors such as aging and the presence of one or more comorbidities, which are intriguingly characterized also by unhealthy microbiome status. Moreover, these comorbidities require complex pharmacological regimens known as "polypharmacy" that may further affect microbiota integrity and worsen the resilience to viral infections. This complex situation may represent a further and underestimated risk with regard to COVID-19 clinical burden for the elderly and comorbid people. Here, we discuss the possible biological, physiopathological, and clinical implications of gut microbiota in COVID-19 and the strategies to improve/maintain its healthy status as a simple and adjunctive strategy to reduce COVID-19 virulence and socio-sanitary burden.
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http://dx.doi.org/10.3389/fcimb.2020.576551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725702PMC
December 2020

Dietary Habits and Psychological States during COVID-19 Home Isolation in Italian College Students: The Role of Physical Exercise.

Nutrients 2020 Nov 28;12(12). Epub 2020 Nov 28.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, 61029 Urbino, Italy.

Social isolation has adverse effects on mental health, physical exercise, and dietary habits. This longitudinal observational study aimed to investigate the effects of mood states and exercise on nutritional choices, on 176 college students (92 males, 84 females; 23 ± 4 years old) during the COVID-19 lockdown. During 21 days, nutrition and exercise were daily monitored, and the mood states assessed. A factor analysis was used to reduce the number of nutritional variables collected. The relationships between exercise, mood and nutrition were investigated using a multivariate general linear model and a mediation model. Seven factors were found, reflecting different nutritional choices. Exercise was positively associated with fruit, vegetables and fish consumption ( = 0.004). Depression and quality of life were, directly and inversely, associated with cereals, legumes ( = 0.005; = 0.004) and low-fat meat intake ( = 0.040; = 0.004). Exercise mediated the effect of mood states on fruit, vegetables and fish consumption, respectively, accounting for 4.2% and 1.8% of the total variance. Poorer mood states possibly led to unhealthy dietary habits, which can themselves be linked to negative mood levels. Exercise led to healthier nutritional choices, and mediating the effects of mood states, it might represent a key measure in uncommon situations, such as home-confinement.
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http://dx.doi.org/10.3390/nu12123660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7759990PMC
November 2020

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

J Neurochem 2021 04 5;157(2):297-311. Epub 2021 Feb 5.

CIBIO, Università degli Studi di Trento, Italy & Dulbecco Telethon Institute, Trento, Italy.

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.
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http://dx.doi.org/10.1111/jnc.15240DOI Listing
April 2021

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

J Neurosci Methods 2021 01 25;347:108960. Epub 2020 Sep 25.

Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", National Research Council, Naples, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jneumeth.2020.108960DOI Listing
January 2021

Depression-Associated Gene Pathway Is Altered by Antidepressant Treatment.

Cells 2020 07 31;9(8). Epub 2020 Jul 31.

Department of Cellular, Computational and Integrative Biology, University of Trento, 38123 Trento, Italy.

The gene has been significantly associated with major depression in genetic studies. 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 pathway in Flinders sensitive line (FSL) rats, in a corticosterone-treated mouse model of depression, and in mouse primary neurons. and were the genes mostly affected by antidepressant treatment, and in opposite directions. 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. 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 expression in hippocampal cultures. expression was increased by nortriptyline in the hypothalamus of FSL rats and in hippocampal neurons. , another IgLON family protein, increased in mouse dentate gyrus after fluoxetine treatment. These findings suggest that 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.
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http://dx.doi.org/10.3390/cells9081818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464991PMC
July 2020

HRR and V˙O2R Fractions Are Not Equivalent: Is It Time to Rethink Aerobic Exercise Prescription Methods?

Med Sci Sports Exerc 2021 01;53(1):174-182

Department of Biomolecular Sciences, Division of Exercise and Health Sciences, University of Urbino Carlo Bo, Urbino, ITALY.

Introduction: According to current guidelines, the intensity of health-enhancing aerobic exercise should be prescribed using a percentage of heart rate reserve (%HRR), which is considered to be more closely associated (showing a 1:1 relation) with the percentage of oxygen uptake reserve (%V˙O2R) rather than with the percentage of maximal oxygen uptake (%V˙O2max) during incremental exercise. However, the associations between %HRR and %V˙O2R and between %HRR and %V˙O2max are under debate; hence, their actual relationships were investigated in this study.

Methods: Data from each stage of a maximal incremental exercise test performed by 737 healthy and physically inactive participants of the HERITAGE Family Study were screened and filtered then used to calculate the individual linear regressions between %HRR and either %V˙O2R or %V˙O2max. For each relationship, the mean slope and intercept of the individual linear regression were compared with 1 and 0 (i.e., the identity line), respectively, using one-sample t-tests. The individual root mean square errors of the actual versus the 1:1 predicted %HRR were calculated for both relationships and compared using a paired-sample t-test.

Results: The mean slopes (%HRR-%V˙O2R, 0.972 ± 0.189; %HRR-%V˙O2max, 1.096 ± 0.216) and intercepts (%HRR-%V˙O2R, 8.855 ± 16.022; %HRR-%V˙O2max, -3.616 ± 18.993) of both relationships were significantly different from 1 and 0, respectively, with high interindividual variability. The average root mean square errors were high and revealed that the %HRR-%V˙O2max relationship was more similar to the identity line (P < 0.001) than the %HRR-%V˙O2R relationship (7.78% ± 4.49% vs 9.25% ± 5.54%).

Conclusions: Because both relationships are different from the identity line and using a single equation may not be appropriate to predict exercise intensity at the individual level, a rethinking of the relationships between the intensity variables may be necessary to ensure that the most suitable health-enhancing aerobic exercise intensity is prescribed.
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http://dx.doi.org/10.1249/MSS.0000000000002434DOI Listing
January 2021

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

Cell Death Discov 2020 8;6:45. Epub 2020 Jun 8.

Institute for Biomedicine, Eurac Research, Affiliated Institute of the University of Lübeck - Via Galvani 31, 39100 Bolzano, Italy.

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.
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http://dx.doi.org/10.1038/s41420-020-0279-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7280235PMC
June 2020

High-intensity Interval Training Promotes the Shift to a Health-Supporting Dietary Pattern in Young Adults.

Nutrients 2020 Mar 21;12(3). Epub 2020 Mar 21.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, 61029 (PU) Urbino, Italy.

A healthy lifestyle is based on a correct diet and regular exercise. Little is known about the effect of different types of exercise on dietary preferences. To address the question of whether high-intensity interval training (HIIT) could modulate spontaneous food choices, an experimental study was carried out on 32 young, healthy normal-weight subjects. The spontaneous diet of each subject has been monitored over nine weeks of indoor-cycling training, divided into three mesocycles with an incremental pattern: total energy intake, macronutrients and micronutrients have been analysed. A two-way mixed model has been used to assess differences in dietary variables; a principal factor analysis has been performed to identify sample subgroups. An increased energy intake (+17.8% at T3; < 0.01) has been observed, although macronutrients' proportions did not vary over time, without differences between sexes. An increase of free fat mass was found in the last mesocycle (+3.8%), without an augmentation of body weight, when, despite the increased training load, a stabilization of energy intake occurred. Three different subgroups characterized by different dietary modifications could be identified among participants that showed a common trend towards a healthier diet. Nine weeks of HIIT promoted a spontaneous modulation of food choices and regulation of dietary intake in young normal-weight subjects aged 21-24. Importantly, this life-period is critical to lay the foundation of correct lifestyles to prevent metabolic diseases and secure a healthy future with advancing age.
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http://dx.doi.org/10.3390/nu12030843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146399PMC
March 2020

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

Sci Rep 2020 03 2;10(1):3799. Epub 2020 Mar 2.

CIBIO, Università degli Studi di Trento, Italy & Dulbecco Telethon Institute, Trento, Italy.

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.
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http://dx.doi.org/10.1038/s41598-020-60834-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052203PMC
March 2020

Effects of a commercially available branched-chain amino acid-alanine-carbohydrate-based sports supplement on perceived exertion and performance in high intensity endurance cycling tests.

J Int Soc Sports Nutr 2020 Jan 20;17(1). Epub 2020 Jan 20.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino, Italy.

Background: Sports nutritional supplements containing branched-chain amino acids (BCAA) have been widely reported to improve psychological and biological aspects connected to central fatigue and performance in endurance exercise, although the topic is still open to debate. The aim of the present study was to determine whether the intake of a commercially available BCAA-based supplement, taken according to the manufacturer's recommendations, could affect the rating of perceived exertion (RPE) and performance indexes at the beginning (1d) and end of a 9-week (9w) scheduled high intensity interval training program, with an experimental approach integrating the determination of psychometric, performance, metabolic and blood biochemical parameters.

Methods: This was a randomized double-blind placebo-controlled study. Thirty-two untrained, healthy young adults (20 males and 12 female) were enrolled. A high-intensity endurance cycling (HIEC) test was used to induce fatigue in the participants: HIEC consisted in ten 90 s sprints interspersed by ten 3 min recovery phases and followed by a final step time to exhaustion was used. In parallel with RPE, haematological values (creatine kinase, alanine, BCAA, tryptophan, ammonia and glucose levels), and performance indexes (maximal oxygen consumption - VO, power associated with lactate thresholds - W, W and time to exhaustion - TTE) were assessed. All subject took the supplement (13.2 g of carbohydrates; 3.2 g of BCAA and 1.6 g of L-alanine per dose) or placebo before each test and training session. Dietary habits and training load were monitored during the entire training period.

Results: The administration of the supplement (SU) at 1d reduced RPE by 9% during the recovery phase, as compared to the placebo (PL); at 9w the RPE scores were reduced by 13 and 21% during the sprint and recovery phase, respectively; at 9w, prolonged supplement intake also improved TTE and TRIMP. SU intake invariably promoted a rapid increase (within 1 h) of BCAA serum blood levels and prevented the post-HIEC tryptophan: BCAA ratio increase found in the PL group, at both 1d and 9w. There was no difference in dietary habits between groups and those habits did not change over time; no difference in glycemia was found between SU and PL. VO, W and W values improved over time, but were unaffected by supplement intake.

Conclusions: On the whole, these results suggest that i) the intake of the BCAA-based commercially available supplement used in this study reduces RPE as a likely consequence of an improvement in the serum tryptophan: BCAA ratio; ii) over time, reduced RPE allows subjects to sustain higher workloads, leading to increased TRIMP and TTE.
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http://dx.doi.org/10.1186/s12970-020-0337-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6971972PMC
January 2020

Mutual Interactions among Exercise, Sport Supplements and Microbiota.

Nutrients 2019 Dec 20;12(1). Epub 2019 Dec 20.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, 61029 (PU) Urbino, Italy.

The adult gut microbiota contains trillions of microorganisms of thousands of different species. Only one third of gut microbiota are common to most people; the rest are specific and contribute to enhancing genetic variation. Gut microorganisms significantly affect host nutrition, metabolic function, immune system, and redox levels, and may be modulated by several environmental conditions, including physical activity and exercise. Microbiota also act like an endocrine organ and is sensitive to the homeostatic and physiological changes associated with training; in turn, exercise has been demonstrated to increase microbiota diversity, consequently improving the metabolic profile and immunological responses. On the other side, adaptation to exercise might be influenced by the individual gut microbiota that regulates the energetic balance and participates to the control of inflammatory, redox, and hydration status. Intense endurance exercise causes physiological and biochemical demands, and requires adequate measures to counteract oxidative stress, intestinal permeability, electrolyte imbalance, glycogen depletion, frequent upper respiratory tract infections, systemic inflammation and immune responses. Microbiota could be an important tool to improve overall general health, performance, and energy availability while controlling inflammation and redox levels in endurance athletes. The relationship among gut microbiota, general health, training adaptation and performance, along with a focus on sport supplements which are known to exert some influence on the microbiota, will be discussed.
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http://dx.doi.org/10.3390/nu12010017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019274PMC
December 2019

Levetiracetam treatment ameliorates LRRK2 pathological mutant phenotype.

J Cell Mol Med 2019 12 27;23(12):8505-8510. Epub 2019 Sep 27.

Department of Biomedical Sciences, University of Sassari, Sassari, Italy.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). The LRRK2 physiological and pathological function is still debated. However, different experimental evidence based on LRRK2 cellular localization and LRRK2 protein interactors suggests that LRRK2 may be part and regulate a protein network modulating vesicle dynamics/trafficking. Interestingly, the synaptic vesicle protein SV2A is part of this protein complex. Importantly, SV2A is the binding site of the levetiracetam (LEV), a compound largely used in human therapy for epilepsy treatment. The binding of LEV to SV2A reduces the neuronal firing by the modulation of vesicle trafficking although by an unclear molecular mechanism. In this short communication, we have analysed the interaction between the LRRK2 and SV2A pathways by LEV treatment. Interestingly, LEV significantly counteracts the effect of LRRK2 G2019S pathological mutant expression in three different cellular experimental models. Our data strongly suggest that LEV treatment may have a neuroprotective effect on LRRK2 pathological mutant toxicity and that LEV repositioning could be a viable compound for PD treatment.
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http://dx.doi.org/10.1111/jcmm.14674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850958PMC
December 2019

Nanolipid-Trehalose Conjugates and Nano-Assemblies as Putative Autophagy Inducers.

Pharmaceutics 2019 Aug 20;11(8). Epub 2019 Aug 20.

Dipartimento di Chimica, Università degli Studi di Milano, Via Golgi 19, 20133 Milano, Italy.

The disaccharide trehalose is an autophagy inducer, but its pharmacological application is severely limited by its poor pharmacokinetics properties. Thus, trehalose was coupled via suitable spacers with squalene (in 1:2 and 1:1 stoichiometry) and with betulinic acid (1:2 stoichiometry), in order to yield the corresponding nanolipid-trehalose conjugates , and . The conjugates were assembled to produce the corresponding nano-assemblies (NAs) , and . The synthetic and assembly protocols are described in detail. The resulting NAs were characterized in terms of loading and structure, and tested in vitro for their capability to induce autophagy. Our results are presented and thoroughly commented upon.
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http://dx.doi.org/10.3390/pharmaceutics11080422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723367PMC
August 2019

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

J Cell Sci 2019 09 26;132(18). Epub 2019 Sep 26.

Laboratory of Molecular and Cellular Neurobiology, Department of Cellular, Computational and Integrative Biology-CIBIO, University of Trento, 38123 Trento, Italy

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.
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http://dx.doi.org/10.1242/jcs.222802DOI Listing
September 2019

Neurostore: A Novel Cryopreserving Medium for Primary Neurons.

Bio Protoc 2019 Jun 20;9(12):e3270. Epub 2019 Jun 20.

CIBIO, Dulbecco Telethon Institute, University of Trento, Trento, Italy.

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 studies and significantly reduces the sacrifice of animals. Here we describe that Neurostore buffer supports the cryopreservation of primary neurons.
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http://dx.doi.org/10.21769/BioProtoc.3270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854206PMC
June 2019

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

J Neurochem 2019 08 10;150(3):264-281. Epub 2019 Jul 10.

Department of Experimental Medicine, University of Genova, Genova, Italy.

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.
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http://dx.doi.org/10.1111/jnc.14778DOI Listing
August 2019

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

Int J Mol Sci 2019 Feb 11;20(3). Epub 2019 Feb 11.

Institute for Biomedicine, Eurac Research, Affiliated Institute of the University of Lübeck, Via Galvani 31, 39100 Bolzano, Italy.

Mutations in the 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.
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http://dx.doi.org/10.3390/ijms20030748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386878PMC
February 2019

Muscle and Bone Health in Postmenopausal Women: Role of Protein and Vitamin D Supplementation Combined with Exercise Training.

Nutrients 2018 08 16;10(8). Epub 2018 Aug 16.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, Urbino 61029 (PU), Italy.

Menopause is an age-dependent physiological condition associated with a natural decline in oestrogen levels, which causes a progressive decrease of muscle mass and strength and bone density. Sarcopenia and osteoporosis often coexist in elderly people, with a prevalence of the latter in elderly women. The profound interaction between muscle and bone induces a negative resonance between the two tissues affected by these disorders worsening the quality of life in the postmenopausal period. It has been estimated that at least 1 in 3 women over age 50 will experience osteoporotic fractures, often requiring hospitalisation and long-term care, causing a large financial burden to health insurance systems. Hormonal replacement therapy is effective in osteoporosis prevention, but concerns have been raised with regard to its safety. On the whole, the increase in life expectancy for postmenopausal women along with the need to improve their quality of life makes it necessary to develop specific and safe therapeutic strategies, alternative to hormonal replacement therapy, targeting both sarcopenia and osteoporosis progression. This review will examine the rationale and the effects of dietary protein, vitamin D and calcium supplementation combined with a specifically-designed exercise training prescription as a strategy to counteract these postmenopausal-associated disorders.
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http://dx.doi.org/10.3390/nu10081103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116194PMC
August 2018

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

Brain 2018 09;141(9):2772-2794

Local Micro-environment and Brain Development Laboratory, Italian Institute of Technology, Genoa, Italy.

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.
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http://dx.doi.org/10.1093/brain/awy190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113639PMC
September 2018

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

Front Cell Neurosci 2018 22;12:81. Epub 2018 Mar 22.

CIBIO, Dulbecco Telethon Institute, University of Trento, Trento, Italy.

Primary neuronal culture from rodents is a well-established model to investigate cellular neurobiology . 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.
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http://dx.doi.org/10.3389/fncel.2018.00081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874515PMC
March 2018

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

Front Mol Neurosci 2018 28;11:64. Epub 2018 Feb 28.

Dulbecco Telethon Institute, CIBIO, Università degli Studi di Trento, Trento, Italy.

Mutations in leucine-rich repeat kinase 2 gene () 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.
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http://dx.doi.org/10.3389/fnmol.2018.00064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835904PMC
February 2018

PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2.

Front Mol Neurosci 2017 14;10:417. Epub 2017 Dec 14.

Department of Biology, University of Padova, Padova, Italy.

Mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD) and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1) Activated Kinase 6 (PAK6). Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.
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http://dx.doi.org/10.3389/fnmol.2017.00417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735978PMC
December 2017

Bacteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles (Tuber borchii).

Appl Microbiol Biotechnol 2018 Feb 30;102(3):1429-1441. Epub 2017 Nov 30.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, via Saffi 2, 61029, Urbino, Italy.

Iron exopolysaccharide nanoparticles were biogenerated during ferric citrate fermentation by Klebsiella oxytoca DSM 29614. Before investigating their effects on Tuber borchii ("bianchetto" truffle) mycelium growth and morphology, they were tested on human K562 cell line and Lentinula edodes pure culture and shown to be non-toxic. Using these nanoparticles as iron supplement, the truffles showed extremely efficient iron uptake of over 300 times that of a commercial product. This avoided morphological changes in T. borchii due to lack of iron during growth and, with optimum nanoparticle dosage, increased growth without cell wall disruption or alteration of protoplasmatic hyphal content, the nuclei, mitochondria, and rough endoplasmic reticula being preserved. No significant modifications in gene expression were observed. These advantages derive from the completely different mechanism of iron delivery to mycelia compared to commercial iron supplements. The present data, in fact, show the nanoparticles attached to the cell wall, then penetrating it non-destructively without damage to cell membrane, mitochondria, chromatin, or ribosome. Low dosage significantly improved mycelium growth, without affecting hyphal morphology. Increases in hyphal diameter and septal distance indicated a healthier state of the mycelia compared to those grown in the absence of iron or with a commercial iron supplement. These positive effects were confirmed by measuring fungal biomass as mycelium dry weight, total protein, and ergosterol content. This "green" method for biogenerating iron exopolysaccharide nanoparticles offers many advantages, including significant economic savings, without toxic effects on the ectomycorrhizal fungus, opening the possibility of using them as iron supplements in truffle plantations.
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http://dx.doi.org/10.1007/s00253-017-8615-8DOI Listing
February 2018

Extracellular Vesicles Released by Oxidatively Injured or Intact C2C12 Myotubes Promote Distinct Responses Converging toward Myogenesis.

Int J Mol Sci 2017 Nov 22;18(11). Epub 2017 Nov 22.

Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino, Italy.

Myogenic differentiation is triggered, among other situations, in response to muscle damage for regenerative purposes. It has been shown that during myogenic differentiation, myotubes release extracellular vesicles (EVs) which participate in the signalling pattern of the microenvironment. Here we investigated whether EVs released by myotubes exposed or not to mild oxidative stress modulate the behaviour of targeted differentiating myoblasts and macrophages to promote myogenesis. We found that EVs released by oxidatively challenged myotubes (H₂O₂-EVs) are characterized by an increased loading of nucleic acids, mainly DNA. In addition, incubation of myoblasts with H₂O₂-EVs resulted in a significant decrease of myotube diameter, myogenin mRNA levels and myosin heavy chain expression along with an upregulation of proliferating cell nuclear antigen: these effects collectively lead to an increase of recipient myoblast proliferation. Notably, the EVs from untreated myotubes induced an opposite trend in myoblasts, that is, a slight pro-differentiation effect. Finally, H₂O₂-EVs were capable of eliciting an increased interleukin 6 mRNA expression in RAW264.7 macrophages. Notably, this is the first demonstration that myotubes communicate with surrounding macrophages via EV release. Collectively, the data reported herein suggest that myotubes, depending on their conditions, release EVs carrying differential signals which could contribute to finely and coherently orchestrate the muscle regeneration process.
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http://dx.doi.org/10.3390/ijms18112488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713454PMC
November 2017

Concurrent Aerobic and Resistance Training Has Anti-Inflammatory Effects and Increases Both Plasma and Leukocyte Levels of IGF-1 in Late Middle-Aged Type 2 Diabetic Patients.

Oxid Med Cell Longev 2017 21;2017:3937842. Epub 2017 Jun 21.

Department of Biomolecular Sciences, Division of Exercise and Health Sciences, University of Urbino Carlo Bo, Urbino, Italy.

Type 2 diabetes (T2D) is an age-related chronic disease associated with metabolic dysregulation, chronic inflammation, and activation of peripheral blood mononuclear cells (PBMC). The aim of this study was to assess the effects of a concurrent exercise training program on inflammatory status and metabolic parameters of T2D patients. Sixteen male patients (age range 55-70) were randomly assigned to an intervention group ( = 8), which underwent a concurrent aerobic and resistance training program (3 times a week; 16 weeks), or to a control group, which followed physicians' usual diabetes care advices. Training intervention significantly improved patients' body composition, blood pressure, total cholesterol, and overall fitness level. After training, plasma levels of adipokines leptin (-33.9%) and RBP4 (-21.3%), and proinflammatory markers IL-6 (-25.3%), TNF- (-19.8%) and MCP-1 (-15.3%) decreased, whereas anabolic hormone IGF-1 level increased (+16.4%). All improvements were significantly greater than those of control patients. Plasma proteomic profile of exercised patients showed a reduction of immunoglobulin K light chain and fibrinogen as well. Training also induced a modulation of , , and mRNAs in the PBMCs. These findings confirm that concurrent aerobic and resistance training improves T2D-related metabolic abnormalities and has the potential to reduce the deleterious health effects of diabetes-related inflammation.
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http://dx.doi.org/10.1155/2017/3937842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497609PMC
April 2018

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

Sci Rep 2017 07 14;7(1):5377. Epub 2017 Jul 14.

CIBIO, Università degli Studi di Trento, Italy & Dulbecco Telethon Institute, Trento, Italy.

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 β3-Cav2.1 at N-terminus part and β-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.
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http://dx.doi.org/10.1038/s41598-017-05760-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5511190PMC
July 2017

LRRK2 Regulates Voltage-Gated Calcium Channel Function.

Front Mol Neurosci 2016 23;9:35. Epub 2016 May 23.

Department of Physiology, University of Otago Dunedin, New Zealand.

Voltage-gated Ca(2+) (CaV) channels enable Ca(2+) influx in response to membrane depolarization. CaV2.1 channels are localized to the presynaptic membrane of many types of neurons where they are involved in triggering neurotransmitter release. Several signaling proteins have been identified as important CaV2.1 regulators including protein kinases, G-proteins and Ca(2+) binding proteins. Recently, we discovered that leucine rich repeat kinase 2 (LRRK2), a protein associated with inherited Parkinson's disease, interacts with specific synaptic proteins and influences synaptic transmission. Since synaptic proteins functionally interact with CaV2.1 channels and synaptic transmission is triggered by Ca(2+) entry via CaV2.1, we investigated whether LRRK2 could impact CaV2.1 channel function. CaV2.1 channel properties were measured using whole cell patch clamp electrophysiology in HEK293 cells transfected with CaV2.1 subunits and various LRRK2 constructs. Our results demonstrate that both wild type (wt) LRRK2 and the G2019S LRRK2 mutant caused a significant increase in whole cell Ca(2+) current density compared to cells expressing only the CaV2.1 channel complex. In addition, LRRK2 expression caused a significant hyperpolarizing shift in voltage-dependent activation while having no significant effect on inactivation properties. These functional changes in CaV2.1 activity are likely due to a direct action of LRRK2 as we detected a physical interaction between LRRK2 and the β3 CaV channel subunit via coimmunoprecipitation. Furthermore, effects on CaV2.1 channel function are dependent on LRRK2 kinase activity as these could be reversed via treatment with a LRRK2 inhibitor. Interestingly, LRRK2 also augmented endogenous voltage-gated Ca(2+) channel function in PC12 cells suggesting other CaV channels could also be regulated by LRRK2. Overall, our findings support a novel physiological role for LRRK2 in regulating CaV2.1 function that could have implications for how mutations in LRRK2 contribute to Parkinson's disease pathophysiology.
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http://dx.doi.org/10.3389/fnmol.2016.00035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4876133PMC
May 2016