Publications by authors named "Angelo Quattrini"

108 Publications

Nerve pathology in animal models of neuropathies.

J Peripher Nerv Syst 2021 Sep 9. Epub 2021 Sep 9.

Experimental Neurology Unit, School of Medicine and Surgery, University of Milano-Bicocca, Monza, Italy.

To understand the pathology of axonal degeneration and demyelination in peripheral neuropathy, histological investigations in different animal models that mimic some aspects of human peripheral neuropathy are needed. Thus, in the following section of this special issue, the main pathological features of experimental autoimmune neuritis, animal models of chemotherapy-induced peripheral neuropath and of human inherited peripheral neuropathies (IPNs) will be illustrated. When possible, micrographs from animal models and selected human biopsy will be shown side by side.
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http://dx.doi.org/10.1111/jns.12463DOI Listing
September 2021

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis.

JAMA Neurol 2021 Aug 30. Epub 2021 Aug 30.

Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, United Kingdom.

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.

Objective: To identify the genetic variants associated with juvenile ALS.

Design, Setting, And Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.

Main Outcomes And Measures: De novo variants present only in the index case and not in unaffected family members.

Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.

Conclusions And Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.
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http://dx.doi.org/10.1001/jamaneurol.2021.2598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406220PMC
August 2021

Femtomolar Biodetection by a Compact Core-Shell 3D Chiral Metamaterial.

Nano Lett 2021 07 12;21(14):6179-6187. Epub 2021 Jul 12.

CNR NANOTEC Institute of Nanotechnology, Via Monteroni, 73100 Lecce, Italy.

Advanced sensing tools, detecting extremely low concentrations of circulating biomarkers, can open unexplored routes toward early diagnostics and diseases progression monitoring. Here, we demonstrate the sensing capabilities of a chip-based metamaterial, combining 3D chiral geometry with a functional core-shell nanoarchitecture. The chiral metamaterial provides a circular polarization-dependent optical response, allowing analysis in a complex environment without significant background interferences. The functional nanoarchitecture, based on the conformal coating with a polymer shell, modifies the chiral metamaterial near- and far-field optical response because of the energy transfer between dielectric shell polarization charges and plasmonic core free electrons, leading to efficient interaction with biomolecules. The system sensitivity slope is 27 nm/pM, in the detection of TAR DNA-binding protein 43, clinically relevant for neurodegenerative diseases. Measurements were performed in spiked solution and in human serum with concentrations from 1 pM down to 10 fM, which is a range not accessible with common immunological assays, opening new perspectives for next-generation biomedical systems.
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http://dx.doi.org/10.1021/acs.nanolett.1c01791DOI Listing
July 2021

Dysregulated copper transport in multiple sclerosis may cause demyelination via astrocytes.

Proc Natl Acad Sci U S A 2021 Jul;118(27)

Division of Neuroscience, Institute of Experimental Neurology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) San Raffaele Scientific Institute, 20132, Milan, Italy;

Demyelination is a key pathogenic feature of multiple sclerosis (MS). Here, we evaluated the astrocyte contribution to myelin loss and focused on the neurotrophin receptor TrkB, whose up-regulation on the astrocyte finely demarcated chronic demyelinated areas in MS and was paralleled by neurotrophin loss. Mice lacking astrocyte TrkB were resistant to demyelination induced by autoimmune or toxic insults, demonstrating that TrkB signaling in astrocytes fostered oligodendrocyte damage. In vitro and ex vivo approaches highlighted that astrocyte TrkB supported scar formation and glia proliferation even in the absence of neurotrophin binding, indicating TrkB transactivation in response to inflammatory or toxic mediators. Notably, our neuropathological studies demonstrated copper dysregulation in MS and model lesions and TrkB-dependent expression of copper transporter (CTR1) on glia cells during neuroinflammation. In vitro experiments evidenced that TrkB was critical for the generation of glial intracellular calcium flux and CTR1 up-regulation induced by stimuli distinct from neurotrophins. These events led to copper uptake and release by the astrocyte, and in turn resulted in oligodendrocyte loss. Collectively, these data demonstrate a pathogenic demyelination mechanism via the astrocyte release of copper and open up the possibility of restoring copper homeostasis in the white matter as a therapeutic target in MS.
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http://dx.doi.org/10.1073/pnas.2025804118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8271600PMC
July 2021

Loss of function MPZ mutation causes milder CMT1B neuropathy.

J Peripher Nerv Syst 2021 Jun 15;26(2):177-183. Epub 2021 May 15.

Institute of Experimental Neurology (InSpe) and Division of Neuroscience, IRCCS Ospedale San Raffaele, Milano, Italy.

Mutations in Myelin Protein Zero (MPZ) cause CMT1B, the second leading cause of CMT1. Many of the >200 mutations cause neuropathy through a toxic gain of function by the mutant protein such as ER retention, activation of the Unfolded Protein Response (UPR) or disruption of myelin compaction. While there is extensive literature on the loss of function consequences of MPZ in heterozygous Mpz +/- null mice, there is little known of the consequences of MPZ haploinsufficiency in humans. We identified six patients from different families with p.Tyr68Ter or p.Asp104fs heterozygous mutations of MPZ that are predicted to cause a premature termination and nonsense mediated decay of the mutant allele. Five patients were evaluated in Milan and one in Iowa City; all should be haploinsufficient for MPZ. Patients were evaluated clinically and by electrophysiology. Sensory ataxia dominated the clinical presentation with only mild weakness present in five of the six patients. Symptoms presented in adulthood in all patients and only one individual had a CMTNSv2 >5. Deep tendon reflexes were absent in all patients. Patients with likely MPZ loss of function due to mutations that cause haplodeficiency in MPZ have a mild, predominantly large fiber sensory neuropathy that serves as a human equivalent to the neuropathy observed in heterozygous Mpz null mice. Successful therapeutic approaches in treating Mpz deficient mice may be candidates for trials in these and similar patients.
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http://dx.doi.org/10.1111/jns.12452DOI Listing
June 2021

Hemiplegic-type ALS shows a strong correlation between upper, lower motor neuron degeneration and pTDP-43 pathology.

J Neurol Neurosurg Psychiatry 2021 Sep 21;92(9):917. Epub 2021 Apr 21.

Department of Neurology, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Hospital Institute of Experimental Neurology, Milano, Lombardia, Italy.

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http://dx.doi.org/10.1136/jnnp-2021-326512DOI Listing
September 2021

A microfabricated multi-compartment device for neuron and Schwann cell differentiation.

Sci Rep 2021 03 29;11(1):7019. Epub 2021 Mar 29.

CNR NANOTEC - Institute of Nanotechnology, Campus Ecotekne, via Monteroni, 73100, Lecce, Italy.

Understanding the complex communication between different cell populations and their interaction with the microenvironment in the central and peripheral nervous systems is fundamental in neuroscience research. The development of appropriate in vitro approaches and tools, able to selectively analyze and/or probe specific cells and cell portions (e.g., axons and cell bodies in neurons), driving their differentiation into specific cell phenotypes, has become therefore crucial in this direction. Here we report a multi-compartment microfluidic device where up to three different cell populations can be cultured in a fluidically independent circuit. The device allows cell migration across the compartments and their differentiation. We showed that an accurate choice of the device geometrical features and cell culture parameters allows to (1) maximize cell adhesion and proliferation of neuron-like human cells (SH-SY5Y cells), (2) control the inter-compartment cell migration of neuron and Schwann cells, (3) perform long-term cell culture studies in which both SH-SY5Y cells and primary rat Schwann cells can be differentiated towards specific phenotypes. These results can lead to a plethora of in vitro co-culture studies in the neuroscience research field, where tuning and investigating cell-cell and cell-microenvironment interactions are essential.
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http://dx.doi.org/10.1038/s41598-021-86300-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007719PMC
March 2021

Current application of neurofilaments in amyotrophic lateral sclerosis and future perspectives.

Neural Regen Res 2021 Oct;16(10):1985-1991

Neuropathology Unit, Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute; Neurology and Neurorehabilitation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Motor neuron disease includes a heterogeneous group of relentless progressive neurological disorders defined and characterized by the degeneration of motor neurons. Amyotrophic lateral sclerosis is the most common and aggressive form of motor neuron disease with no effective treatment so far. Unfortunately, diagnostic and prognostic biomarkers are lacking in clinical practice. Neurofilaments are fundamental structural components of the axons and neurofilament light chain and phosphorylated neurofilament heavy chain can be measured in both cerebrospinal fluid and serum. Neurofilament light chain and phosphorylated neurofilament heavy chain levels are elevated in amyotrophic lateral sclerosis, reflecting the extensive damage of motor neurons and axons. Hence, neurofilaments are now increasingly recognized as the most promising candidate biomarker in amyotrophic lateral sclerosis. The potential usefulness of neurofilaments regards various aspects, including diagnosis, prognosis, patient stratification in clinical trials and evaluation of treatment response. In this review paper, we review the body of literature about neurofilaments measurement in amyotrophic lateral sclerosis. We also discuss the open issues concerning the use of neurofilaments clinical practice, as no overall guideline exists to date; finally, we address the most recent evidence and future perspectives.
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http://dx.doi.org/10.4103/1673-5374.308072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8343335PMC
October 2021

Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols.

Sci Rep 2021 Feb 11;11(1):3589. Epub 2021 Feb 11.

Division of Neuroscience, Institute of Experimental Neurology, San Raffaele Scientific Institute, Via Olgettina, 60, 20132, Milan, Italy.

Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.
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http://dx.doi.org/10.1038/s41598-021-83241-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878808PMC
February 2021

X-ray phase contrast tomography for the investigation of amyotrophic lateral sclerosis.

J Synchrotron Radiat 2020 Jul 9;27(Pt 4):1042-1048. Epub 2020 Jun 9.

Physics Department `Sapienza' University, CNR-Institute of Nanotechnology, Piazzale Aldo Moro 5, 00185 Rome, Italy.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. Pre-clinical studies drive the development of animal models that well mimic ALS disorder and enable both the dissection of disease processes and an early assessment of therapy efficacy. A comprehensive knowledge of neuronal and vascular lesions in the brain and spinal cord is an essential factor to understand the development of the disease. Spatial resolution and bidimensional imaging are important drawbacks limiting current neuroimaging tools, while neuropathology relies on protocols that may alter tissue chemistry and structure. In contrast, recent ex vivo studies in mice demonstrated that X-ray phase-contrast tomography enables study of the 3D distribution of both vasculature and neuronal networks, without sample sectioning or use of staining. Here we present our findings on ex vivo SOD1 ALS mice spinal cord at a micrometric scale. An unprecedented direct quantification of neuro-vascular alterations at different stages of the disease is shown.
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http://dx.doi.org/10.1107/S1600577520006785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336179PMC
July 2020

Diet, Microbiota and Brain Health: Unraveling the Network Intersecting Metabolism and Neurodegeneration.

Int J Mol Sci 2020 Oct 10;21(20). Epub 2020 Oct 10.

Experimental Neuropathology Unit, Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.

Increasing evidence gives support for the idea that extra-neuronal factors may affect brain physiology and its predisposition to neurodegenerative diseases. Epidemiological and experimental studies show that nutrition and metabolic disorders such as obesity and type 2 diabetes increase the risk of Alzheimer's and Parkinson's diseases after midlife, while the relationship with amyotrophic lateral sclerosis is uncertain, but suggests a protective effect of features of metabolic syndrome. The microbiota has recently emerged as a novel factor engaging strong interactions with neurons and glia, deeply affecting their function and behavior in these diseases. In particular, recent evidence suggested that gut microbes are involved in the seeding of prion-like proteins and their spreading to the central nervous system. Here, we present a comprehensive review of the impact of metabolism, diet and microbiota in neurodegeneration, by affecting simultaneously several aspects of health regarding energy metabolism, immune system and neuronal function. Advancing technologies may allow researchers in the future to improve investigations in these fields, allowing the buildup of population-based preventive interventions and development of targeted therapeutics to halt progressive neurologic disability.
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http://dx.doi.org/10.3390/ijms21207471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590163PMC
October 2020

Impaired flickering of the permeability transition pore causes SPG7 spastic paraplegia.

EBioMedicine 2020 Nov 9;61:103050. Epub 2020 Oct 9.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli-Naples, Italy; Vita-Salute San Raffaele University, Milan, Italy. Electronic address:

Background: Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia type 7 (SPG7), a so-far untreatable degenerative disease of the upper motoneuron with still undefined pathomechanism. The intermittent mitochondrial permeability transition pore (mPTP) opening, called flickering, is an essential process that operates to maintain mitochondrial homeostasis by reducing intra-matrix Ca and reactive oxygen species (ROS) concentration, and is critical for efficient synaptic function.

Methods: We use a fluorescence-based approach to measure mPTP flickering in living cells and biochemical and molecular biology techniques to dissect the pathogenic mechanism of SPG7. In the SPG7 animal model we evaluate the potential improvement of the motor defect, neuroinflammation and neurodegeneration by means of an mPTP inducer, the benzodiazepine Bz-423.

Findings: We demonstrate that paraplegin is required for efficient transient opening of the mPTP, that is impaired in both SPG7 patients-derived fibroblasts and primary neurons from Spg7 mice. We show that dysregulation of mPTP opening at the pre-synaptic terminal impairs neurotransmitter release leading to ineffective synaptic transmission. Lack of paraplegin impairs mPTP flickering by a mechanism involving increased expression and activity of sirtuin3, which promotes deacetylation of cyclophilin D, thus hampering mPTP opening. Pharmacological treatment with Bz-423, which bypasses the activity of CypD, normalizes synaptic transmission and rescues the motor impairment of the SPG7 mouse model.

Interpretation: mPTP targeting opens a new avenue for the potential therapy of this form of spastic paraplegia.

Funding: Telethon Foundation grant (TGMGCSBX16TT); Dept. of Defense, US Army, grant W81XWH-18-1-0001.
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http://dx.doi.org/10.1016/j.ebiom.2020.103050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553352PMC
November 2020

Retromer stabilization results in neuroprotection in a model of Amyotrophic Lateral Sclerosis.

Nat Commun 2020 07 31;11(1):3848. Epub 2020 Jul 31.

INSPE-Institute of Experimental Neurology, San Raffaele Scientific Institute, Milano, Italy.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS.
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http://dx.doi.org/10.1038/s41467-020-17524-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395176PMC
July 2020

From pathogenesis to personalized treatments of neuropathies in hematological malignancies.

J Peripher Nerv Syst 2020 09 7;25(3):212-221. Epub 2020 Aug 7.

Experimental Neuropathology Unit, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.

The peripheral nervous system may be involved at any stage in the course of several hematological diseases, the most common being monoclonal gammopathies (of undetermined significance or malignant) or lymphomas. The underlying pathogenic mechanisms are different and therapies aim at targeting the dangerous either B-cell or plasma cell clones. Recently, high-throughput technologies, and next-generation sequencing have increased our knowledge of hematological diseases pathogenesis by the identification of somatic mutation affecting pivotal signaling pathways. Accordingly, new target therapies are used that may also be borrowed for treatment of neuropathies in hematological diseases.
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http://dx.doi.org/10.1111/jns.12405DOI Listing
September 2020

Neonatal combination therapy improves some of the clinical manifestations in the Mucopolysaccharidosis type I murine model.

Mol Genet Metab 2020 07 11;130(3):197-208. Epub 2020 May 11.

Centro Ricerca M. Tettamanti, Department of Pediatrics, University of Milano-Bicocca, Monza 20900, Italy. Electronic address:

Mucopolysaccharidosis type I (MPS-I), a lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase enzyme, results in the progressive accumulation of glycosaminoglycans and consequent multiorgan dysfunction. Despite the effectiveness of hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy (ERT) in correcting clinical manifestations related to visceral organs, complete improvement of musculoskeletal and neurocognitive defects remains an unmet challenge and provides an impact on patients' quality of life. We tested the therapeutic efficacy of combining HSCT and ERT in the neonatal period. Using a mouse model of MPS-I, we demonstrated that the combination therapy improved clinical manifestations in organs usually refractory to current treatment. Moreover, combination with HSCT prevented the production of anti-IDUA antibodies that negatively impact ERT efficacy. The added benefits of combining both treatments also resulted in a reduction of skeletal anomalies and a trend towards decreased neuroinflammation and metabolic abnormalities. As currently there are limited therapeutic options for MPS-I patients, our findings suggest that the combination of HSCT and ERT during the neonatal period may provide a further step forward in the treatment of this rare disease.
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http://dx.doi.org/10.1016/j.ymgme.2020.05.001DOI Listing
July 2020

Burden of Rare Variants in ALS and Axonal Hereditary Neuropathy Genes Influence Survival in ALS: Insights from a Next Generation Sequencing Study of an Italian ALS Cohort.

Int J Mol Sci 2020 May 8;21(9). Epub 2020 May 8.

Experimental Neuropathology Unit, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, 20132 Milan, Italy.

Although the genetic architecture of amyotrophic lateral sclerosis (ALS) is incompletely understood, recent findings suggest a complex model of inheritance in ALS, which is consistent with a multistep pathogenetic process. Therefore, the aim of our work is to further explore the architecture of ALS using targeted next generation sequencing (NGS) analysis, enriched in motor neuron diseases (MND)-associated genes which are also implicated in axonal hereditary motor neuropathy (HMN), in order to investigate if disease expression, including the progression rate, could be influenced by the combination of multiple rare gene variants. We analyzed 29 genes in an Italian cohort of 83 patients with both familial and sporadic ALS. Overall, we detected 43 rare variants in 17 different genes and found that 43.4% of the ALS patients harbored a variant in at least one of the investigated genes. Of note, 27.9% of the variants were identified in other MND- and HMN-associated genes. Moreover, multiple gene variants were identified in 17% of the patients. The burden of rare variants is associated with reduced survival and with the time to reach King stage 4, i.e., the time to reach the need for percutaneous endoscopic gastrostomy (PEG) positioning or non-invasive mechanical ventilation (NIMV) initiation, independently of known negative prognostic factors. Our data contribute to a better understanding of the molecular basis of ALS supporting the hypothesis that rare variant burden could play a role in the multistep model of disease and could exert a negative prognostic effect. Moreover, we further extend the genetic landscape of ALS to other MND-associated genes traditionally implicated in degenerative diseases of peripheral axons, such as HMN and CMT2.
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http://dx.doi.org/10.3390/ijms21093346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246633PMC
May 2020

Serum phosphorylated neurofilament heavy-chain levels reflect phenotypic heterogeneity and are an independent predictor of survival in motor neuron disease.

J Neurol 2020 Aug 18;267(8):2272-2280. Epub 2020 Apr 18.

Division of Neuroscience, Neuropathology Unit, San Raffaele Scientific Institute, via Olgettina 48, 20132, Milan, Italy.

To investigate the prognostic role and the major determinants of serum phosphorylated neurofilament heavy -chain (pNfH) concentration across a large cohort of motor neuron disease (MND) phenotypes. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum pNfH concentration in 219 MND patients consecutively enrolled in our tertiary MND clinic. A multifactorial analysis was carried out to investigate the major clinical determinants of serum pNfH. Kaplan-Meier survival curves and Cox regression analysis were performed to explore the prognostic value of serum pNfH. Serum pNfH levels were not homogenous among MND phenotypes; higher concentrations in pyramidal, bulbar, and classic phenotypes were observed. C9orf72-MND exhibited higher pNfH concentrations compared to non-C9orf72 MND. Multiple linear regression analysis revealed mean MEP/cMAP and disease progression rate as the two major predictors of serum pNfH levels (R = 0.188; p ≤ 0.001). Kaplan-Meier curves showed a significant difference of survival among MND subgroups when divided into quartiles based on pNfH concentrations, log-rank X = 53.0, p ≤ 0.0001. Our study evidenced that higher serum pNfH concentration is a negative independent prognostic factor for survival. In Cox multivariate model, pNfH concentration showed the highest hazard ratio compared to the other factors influencing survival included in the analysis. pNfH differs among the MND phenotypes and is an independent prognostic factor for survival. This study provides supporting evidence of the role of pNfH as useful prognostic biomarker for MND patients. Neurofilament measurements should be considered in the future prognostic models and in clinical trials for biomarker-based stratification, and to evaluate treatment response.
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http://dx.doi.org/10.1007/s00415-020-09838-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166001PMC
August 2020

Impaired turnover of hyperfused mitochondria in severe axonal neuropathy due to a novel DRP1 mutation.

Hum Mol Genet 2020 01;29(2):177-188

Department of Neurology, IRCCS Ospedale San Raffaele, Milan, Italy.

Mitochondria undergo continuous cycles of fusion and fission in response to physiopathological stimuli. The key player in mitochondrial fission is dynamin-related protein 1 (DRP1), a cytosolic protein encoded by dynamin 1-like (DNM1L) gene, which relocalizes to the outer mitochondrial membrane, where it assembles, oligomerizes and drives mitochondrial division upon guanosine-5'-triphosphate (GTP) hydrolysis. Few DRP1 mutations have been described so far, with patients showing complex and variable phenotype ranging from early death to encephalopathy and/or optic atrophy. The disease is the consequence of defective mitochondrial fission due to faulty DRP1 function. However, the underlying molecular mechanisms and the functional consequences at mitochondrial and cellular level remain elusive. Here we report on a 5-year-old girl presenting psychomotor developmental delay, global hypotonia and severe ataxia due to axonal sensory neuropathy harboring a novel de novo heterozygous missense mutation in the GTPase domain of DRP1 (NM_012062.3:c.436G>A, NP_036192.2: p.D146N variant in DNM1L). Patient's fibroblasts show hyperfused/balloon-like giant mitochondria, highlighting the importance of D146 residue for DRP1 function. This dramatic mitochondrial rearrangement phenocopies what observed overexpressing DRP1-K38A, a well-known experimental dominant negative version of DRP1. In addition, we demonstrated that p.D146N mutation has great impact on peroxisomal shape and function. The p.D146N mutation compromises the GTPase activity without perturbing DRP1 recruitment or assembly, causing decreased mitochondrial and peroxisomal turnover. In conclusion, our findings highlight the importance of sensory neuropathy in the clinical spectrum of DRP1 variants and, for the first time, the impact of DRP1 mutations on mitochondrial turnover and peroxisomal functionality.
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http://dx.doi.org/10.1093/hmg/ddz211DOI Listing
January 2020

Ablation of neuronal ADAM17 impairs oligodendrocyte differentiation and myelination.

Glia 2020 06 18;68(6):1148-1164. Epub 2019 Dec 18.

INSPE, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient propagation of the electric impulse in the nervous system and to maintain neuronal integrity. In the central nervous system (CNS), the development of oligodendrocytes and the process of myelination are regulated by the coordinated action of several positive and negative cell-extrinsic factors. We and others previously showed that secretases regulate the activity of proteins essential for myelination. We now report that the neuronal α-secretase ADAM17 controls oligodendrocyte differentiation and myelin formation in the CNS. Ablation of Adam17 in neurons impairs in vivo and in vitro oligodendrocyte differentiation, delays myelin formation throughout development and results in hypomyelination. Furthermore, we show that this developmental defect is, in part, the result of altered Notch/Jagged 1 signaling. Surprisingly, in vivo conditional loss of Adam17 in immature oligodendrocytes has no effect on myelin formation. Collectively, our data indicate that the neuronal α-secretase ADAM17 is required for proper CNS myelination. Further, our studies confirm that secretases are important post-translational regulators of myelination although the mechanisms controlling CNS and peripheral nervous system (PNS) myelination are distinct.
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http://dx.doi.org/10.1002/glia.23765DOI Listing
June 2020

Prostaglandin D2 synthase modulates macrophage activity and accumulation in injured peripheral nerves.

Glia 2020 01 3;68(1):95-110. Epub 2019 Sep 3.

Division of Neuroscience, INSPE, IRCCS San Raffaele Scientific Institute, Milan, Italy.

We have previously reported that prostaglandin D2 Synthase (L-PGDS) participates in peripheral nervous system (PNS) myelination during development. We now describe the role of L-PGDS in the resolution of PNS injury, similarly to other members of the prostaglandin synthase family, which are important for Wallerian degeneration (WD) and axonal regeneration. Our analyses show that L-PGDS expression is modulated after injury in both sciatic nerves and dorsal root ganglia neurons, indicating that it might play a role in the WD process. Accordingly, our data reveals that L-PGDS regulates macrophages phagocytic activity through a non-cell autonomous mechanism, allowing myelin debris clearance and favoring axonal regeneration and remyelination. In addition, L-PGDS also appear to control macrophages accumulation in injured nerves, possibly by regulating the blood-nerve barrier permeability and SOX2 expression levels in Schwann cells. Collectively, our results suggest that L-PGDS has multiple functions during nerve regeneration and remyelination. Based on the results of this study, we posit that L-PGDS acts as an anti-inflammatory agent in the late phases of WD, and cooperates in the resolution of the inflammatory response. Thus, pharmacological activation of the L-PGDS pathway might prove beneficial in resolving peripheral nerve injury.
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http://dx.doi.org/10.1002/glia.23705DOI Listing
January 2020

Limitations in daily activities and general perception of quality of life: Long term follow-up in patients with anti-myelin-glycoprotein antibody polyneuropathy.

J Peripher Nerv Syst 2019 09 26;24(3):276-282. Epub 2019 Aug 26.

Department of Neurosciences, University of Padova, Padova, Italy.

In this study, we assessed the modifications over time of daily activities and quality of life (QoL) in 32 subjects with anti-myelin-glycoprotein (MAG) antibody neuropathy. A widespread panel including clinical scores and patient-reported questionnaires, in compliance of the terms by the International Classification of Functioning, Disability, and Health (ICF) of the World Health Organization (WHO), was employed at enrollment (T0) and at follow-up evaluation (T1) after a mean interval of 15.4 ± 5.7 months. The Sensory Modality Sum score (SMS) at four limbs showed a significant worsening over time (mean score 27.2 ± 3.9 at T0 vs 25.7 ± 3 at T1 at upper limbs, P = .03; 20.5 ± 4.8 at T0 vs 18.6 ± 5.9 at T1 at lower limbs, P = .04). The Visual Analogue Scale (VAS) for pain significantly worsened at upper limbs at T1 (mean values 0.84 ± 1.95 at T0 vs 1.78 ± 2.6 at T1, P = .03). All the other tests did not show significant differences between T0 and T1. In the subgroup who underwent rituximab (15/32 treated before T0, 3/32 patients treated between T0 and T1 with median interval of 1 year), no significant differences were observed between T0 and T1. Despite the quite long follow-up, statistical significance was not achieved either for the limited number of patients or for the lack of sensitive outcome measures. In our cohort, the significant worsening of the SMS and VAS after a median of 14 months can be considered as a reliable expression of the natural history of the disease, and suggest that these scales might represent possible outcome measures in anti-MAG antibody neuropathy.
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http://dx.doi.org/10.1111/jns.12342DOI Listing
September 2019

The Peripheral Nervous System in Amyotrophic Lateral Sclerosis: Opportunities for Translational Research.

Front Neurosci 2019 25;13:601. Epub 2019 Jun 25.

Experimental Neuropathology Unit, Division of Neuroscience, Institute of Experimental Neurology - San Raffaele Scientific Institute, Milan, Italy.

Although amyotrophic lateral sclerosis (ALS) has been considered as a disorder of the motor neuron (MN) cell body, recent evidences show the non-cell-autonomous pathogenic nature of the disease. Axonal degeneration, loss of peripheral axons and destruction of nerve terminals are early events in the disease pathogenic cascade, anticipating MN degeneration, and the onset of clinical symptoms. Therefore, although ALS and peripheral axonal neuropathies should be differentiated in clinical practice, they also share damage to common molecular pathways, including axonal transport, RNA metabolism and proteostasis. Thus, an extensive evaluation of the molecular events occurring in the peripheral nervous system (PNS) could be fundamental to understand the pathogenic mechanisms of ALS, favoring the discovery of potential disease biomarkers, and new therapeutic targets.
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http://dx.doi.org/10.3389/fnins.2019.00601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603245PMC
June 2019

Distinct Protein Expression Networks are Activated in Microglia Cells after Stimulation with IFN-γ and IL-4.

Cells 2019 06 12;8(6). Epub 2019 Jun 12.

Department of Biological and Environmental Sciences and Technologies, University of Salento, 73100 Lecce, Italy.

Microglia cells are the primary immune population of the central nervous system with a role in the regulation of several physiological and pathological conditions. Upon appropriate stimulation, microglia cells can be polarized in a pro-inflammatory M1-like or anti-inflammatory M2-like status. Biological processes and pathways engaged in microglia polarization are starting to be elucidated. To help clarify this, we used a liquid chromatography-mass spectrometry (LC-MS/MS) label free approach to characterize the proteomic profile of human microglia cell line (CHME-5) stimulated with gamma-interferon (IFN-γ) and interleukin-4 (IL-4) to induce a M1 or M2 phenotype, respectively. Outside the classical M1/M2 polarization markers, the M1 status appears to center around the activation of a classical inflammatory response and through the activation of multiple signaling pathways. M2 polarization resulted in a different pattern of protein modulation related to RNA and cellular metabolic processes. Together, our findings provide information regarding the protein changes specific to M1 and M2 activation states, and potentially link the polarization of microglia cells to the acquisition of a specific proteomic profile.
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http://dx.doi.org/10.3390/cells8060580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628119PMC
June 2019

Neural Stem Cells of the Subventricular Zone Contribute to Neuroprotection of the Corpus Callosum after Cuprizone-Induced Demyelination.

J Neurosci 2019 07 28;39(28):5481-5492. Epub 2019 May 28.

Neuroimmunology Unit, Institute of Experimental Neurology, Division of Neuroscience, IRCCS San Raffaele Hospital, 20132 Milan, Italy,

Myelin loss occurring in demyelinating diseases, including multiple sclerosis, is the leading cause of long-lasting neurological disability in adults. While endogenous remyelination, driven by resident oligodendrocyte precursor cells (OPCs), might partially compensate myelin loss in the early phases of demyelinating disorders, this spontaneous reparative potential fails at later stages. To investigate the cellular mechanisms sustaining endogenous remyelination in demyelinating disorders, we focused our attention on endogenous neural precursor cells (eNPCs) located within the subventricular zone (SVZ) since this latter area is considered one of the primary sources of new OPCs in the adult forebrain. First, we fate mapped SVZ-eNPCs in cuprizone-induced demyelination and found that SVZ endogenous neural stem/precursor cells are recruited during the remyelination phase to the corpus callosum (CC) and are capable of forming new oligodendrocytes. When we ablated SVZ-derived eNPCs during cuprizone-induced demyelination in female mice, the animals displayed reduced numbers of oligodendrocytes within the lesioned CC. Although this reduction in oligodendrocytes did not impact the ensuing remyelination, eNPC-ablated mice experienced increased axonal loss. Our results indicate that, in toxic models of demyelination, SVZ-derived eNPCs contribute to support axonal survival. One of the significant challenges in MS research is to understand the detrimental mechanisms leading to the failure of CNS tissue regeneration during disease progression. One possible explanation is the inability of recruited oligodendrocyte precursor cells (OPCs) to complete remyelination and to sustain axonal survival. The contribution of endogenous neural precursor cells (eNPCs) located in the subventricular zone (SVZ) to generate new OPCs in the lesion site has been debated. Using transgenic mice to fate map and to selectively kill SVZ-derived eNPCs in the cuprizone demyelination model, we observed migration of SVZ-eNPCs after injury and their contribution to oligodendrogenesis and axonal survival. We found that eNPCs are dispensable for remyelination but protect partially from increased axonal loss.
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http://dx.doi.org/10.1523/JNEUROSCI.0227-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616285PMC
July 2019

Concurrence of NMOSD and ALS in a patient with hexanucleotide repeat expansions of C9orf72.

Amyotroph Lateral Scler Frontotemporal Degener 2019 08 22;20(5-6):449-452. Epub 2019 Apr 22.

a Department of Neurology, Institute of Experimental Neurology (INSPE), Division of Neuroscience, IRCCS San Raffaele Scientific Institute , Vita-Salute San Raffaele University , Milan , Italy.

We describe a patient, previously known for NMOSD, who presented a rapidly progressive worsening of muscle strength, respiratory, and bulbar functions. ALS associated with cognitive impairment was diagnosed, while genetic analysis revealed a hexanucleotide repeat expansion in the C9orf72 gene. To the best of our knowledge, this is the first reported C9orf72-ALS patient with concurrent NMOSD. In consideration of the low prevalence of these two diseases, a by-chance co-occurrence is unlikely. Although the discovery of a disease-specific serum AQP4-IgG antibody has led to a broadening of the NMOSD, a progressive neurological deterioration, as shown by our patient, should be considered as a "red flag", leading to alternative diagnostic hypotheses. Our report supports the hypothesis that in C9orf72-ALS neuroinflammation may contribute to disease penetrance or to determine an aggressive clinical phenotype. Further investigations are needed in order to establish possible shared neuroinflammatory patterns between ALS, NMOSD, and other neuroinflammatory disorders.
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http://dx.doi.org/10.1080/21678421.2019.1604761DOI Listing
August 2019

Optic nerve involvement in experimental autoimmune encephalomyelitis to homologous spinal cord homogenate immunization in the dark agouti rat.

J Neuroimmunol 2018 12 25;325:1-9. Epub 2018 Sep 25.

San Raffaele Scientific Institute, via Olgettina 60, 20132 Milan, Italy. Electronic address:

Dark-Agouti rats were immunized with spinal cord homogenate to develop Experimental Autoimmune Encephalomyelitis, a model of multiple sclerosis. We assessed motor signs and recorded VEPs for five or eight weeks with epidural or epidermal electrodes, respectively, with final histopathology of optic nerves (ONs). Injected rats exhibited motor deficits a week after immunization. VEP delays arose from the 2nd to the 5th week, when a recovery occurred in epidermal-recorded rats. ON damage appeared in epidural-, but not in epidermal-recorded rats, probably due to a remyelination process. VEP could be exploited as neurophysiological marker to test novel treatments against neurodegeneration involving ONs.
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http://dx.doi.org/10.1016/j.jneuroim.2018.09.009DOI Listing
December 2018

Functioning and quality of life in patients with neuropathy associated with anti-MAG antibodies.

J Neurol 2018 Dec 10;265(12):2927-2933. Epub 2018 Oct 10.

Division of Neuroscience, Department of Neurology, Institute of Experimental Neurology (INSPE), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Although anti-myelin-associated glycoprotein (MAG) antibody neuropathy is reported as a slowly progressive disease, it can lead to significant disability and impairment of health-related quality of life (HR-QoL) and social participation. The aim of this cross-sectional study was to evaluate the functioning and HR-QoL determinants in 67 patients with anti-MAG neuropathy in terms of the International Classification of Functioning, Disability, and Health (ICF). Evaluations included: Medical Research Council (MRC) sum score, Sensory Modality Sum score (SMS), Berg balance scale (BBS), Fatigue Severity Scale (FSS), Visual Analogue Scale (VAS) for pain, 9-Hole Peg Test (9-HPT), 6-min Walk Distance (6MWD), Impact on Participation and Autonomy (IPA) and the physical component score (PCS) and mental component score (MCS) of the short-form-36 health status scale (SF-36) HR-QoL measure. In the regression models, 6MWD was the most reliable predictor of PCS, explaining the 52% of its variance, while the strongest determinants of 6MWD were BBS and FSS, explaining the 41% of its variance. Consistently, VAS and BBS were good predictor of PCS, explaining together 54% of its variance. FSS was the most reliable determinant of MCS, explaining 25% of its variance. SMS and MRC were not QoL determinants. The results of our study suggest that 6MWD and FSS might be considered as potential meaningful outcome measures in future clinical trials. Furthermore, neurorehabilitation interventions aimed at improving balance and walking performance, fatigue management, and specific pain relief therapy should be considered to ameliorate participation in social life and HR-QoL in anti-MAG neuropathy patients.
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http://dx.doi.org/10.1007/s00415-018-9081-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244677PMC
December 2018

An update on the diagnosis and management of the polyneuropathy of POEMS syndrome.

J Neurol 2019 Jan 27;266(1):258-267. Epub 2018 Sep 27.

Neuropathology Unit, Division of Neuroscience and Department of Neurology, Institute of Experimental Neurology, San Raffaele Scientific Institute, Via Olgettina 48, 20132, Milan, Italy.

POEMS syndrome is a rare, chronic, disabling paraneoplastic disorder characterized by peripheral neuropathy, organomegaly, endocrinopathy, monoclonal plasma cells disorder and skin changes. Diagnosis relies on the fulfillment of a set of clinical criteria of which polyneuropathy and a monoclonal plasma cell dyscrasia are early and essential features. Treatment may be either local or systemic and is aimed at the monoclonal plasma cell disorder. Our knowledge of the pathogenesis underlying the POEMS syndrome has advanced greatly over the past years, favoring an important progression in the recognition and management of this disorder. Here, we discuss the recent literature that has advanced our knowledge of the pathogenesis and clinical management of the polyneuropathy in POEMS syndrome.
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http://dx.doi.org/10.1007/s00415-018-9068-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343023PMC
January 2019

Lab-on-Chip for Exosomes and Microvesicles Detection and Characterization.

Sensors (Basel) 2018 Sep 20;18(10). Epub 2018 Sep 20.

CNR NANOTEC Institute of Nanotechnology, via Monteroni, 73100 Lecce, Italy.

Interest in extracellular vesicles and in particular microvesicles and exosomes, which are constitutively produced by cells, is on the rise for their huge potential as biomarkers in a high number of disorders and pathologies as they are considered as carriers of information among cells, as well as being responsible for the spreading of diseases. Current methods of analysis of microvesicles and exosomes do not fulfill the requirements for their in-depth investigation and the complete exploitation of their diagnostic and prognostic value. Lab-on-chip methods have the potential and capabilities to bridge this gap and the technology is mature enough to provide all the necessary steps for a completely automated analysis of extracellular vesicles in body fluids. In this paper we provide an overview of the biological role of extracellular vesicles, standard biochemical methods of analysis and their limits, and a survey of lab-on-chip methods that are able to meet the needs of a deeper exploitation of these biological entities to drive their use in common clinical practice.
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http://dx.doi.org/10.3390/s18103175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6210978PMC
September 2018
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