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(1) Multiple sclerosis (MS) is identified by a complex interaction between central inflammation and neurodegeneration. Genetic individual variability could play a significative role in clinical presentation. The interleukin-5 (IL-5) rs2069812 single-nucleotide polymorphism (SNP) seems to define the clinical course of Th2 autoimmune diseases, while its role in MS has never been investigated. (2) In a group of 230 patients diagnosed with relapsing-remitting MS (RR-MS) or progressive MS (P-MS) and controls (IC), rs2069812 polymorphism, cerebrospinal fluid (CSF) levels of inflammatory mediators, and clinical and demographic characteristics were determined. In RR-MS patients, No Evidence of Disease Activity (NEDA-3) at three years of follow-up was detected. (3) We identified higher levels of proinflammatory cytokines, particularly IL-2 (median [IQR], RR-MS = 0.2 [0-0.7]; P-MS = 0.1 [0-1.6]; IC = 0.1 [0.0-0.1]; p < 0.005), IL-6 (RR-MS = 0.9 [0.3-2.3]; P-MS = 0.8 [0.1-2.7]; IC = 0.1 [0.0-0.5]; p < 0.005), IL-12 (RR-MS = 0.5 [0-1.1]; P-MS = 0.5 [0-1.1]; IC = 0.0 [0.0-0.3]; p < 0.005), and GM-CSF (RR-MS = 15.6 [4.8-26.4]; P-MS = 14 [3.3-29.7]; IC = 8.9 [4.7-11.7]; p < 0.005) in MS patients compared with IC. Conversely, anti-inflammatory cytokines, specifically IL-5 (RR-MS = 0.65 [0-2.4]; P-MS = 0.1 [0-0.8]; IC = 1.7 [0.6-2.8]; p < 0.005) and IL-1ra (RR-MS = 14.7 [4.9-26.4]; P-MS = 13.1 [4.7-22.2]; IC = 27.8 [17.7-37.6]; p < 0.005) were higher in controls. According to rs2069812, in MS patients, the T-allele was associated with higher concentrations of proinflammatory mediators (IL-2, CT/TT = 0.2 [0.0-2.0]; CC = 0.1 [0.0-0.4], p = 0.015; IL-6, CT/TT = 1.2 [0.4-3.2] vs. CC = 0.7 [0.1-1.7], p = 0.007; IL-15, CT/TT = 0.1 [0.0-9.5] vs. CC = 0.0 [0.0-0.1], p = 0.019; and GM-CSF, CT/TT = 0.1 [0.0-0.6] vs. CC = 0.05 [0.0-0.1], p < 0.001), and CC was associated with anti-inflammatory mediators (IL-5, CT/TT = 0.03 [0.0-1.9] vs. CC = 1.28 [0.0-2.7], p = 0.001; IL-1ra, CT/TT = 12.1 [4.1-25.9] vs. CC = 18.1 [12.1-26.9], p = 0.006). We found the same differences in RR-MS patients (IL-2, T-allele median [IQR] = 0.3 [0.0-2.0] vs. C-allele, median [IQR] = 0.04 [0.0-0.3]; p = 0.005; IL-6, T-allele, median [IQR] = 1.3 [0.4-3.3] vs. C-allele, median [IQR] = 0.6 [0.03-1.5]; p = 0.001; IL-15, T-allele, median [IQR] = 0.1 [0.0-9.5] vs. C-allele, median [IQR] = 0.0 [0.0-0.1]; p = 0.008; GM-CSF, T-allele, median [IQR] = 0.1 [0.0-97.9] vs. C-allele, median [IQR] = 0.0 [0.0-0.001]; p < 0.001; IL-5, T-allele, median [IQR] = 0.02 [0.0-2.2] vs. C-allele, median [IQR] = 1.5 [0.0-2.9]; p = 0.016; and IL-1ra, T-allele, median [IQR] = 12.1 [4.3-26.4] vs. C-allele, median [IQR] = 18.5 [12.7-28.3]; p = 0.006) but not in P-MS, except for IL-5 (T-allele, median [IQR] = 0.1 [0-0.23] vs. C-allele, median [IQR] = 0.6 [0.0-2.5]; p = 0.022). Finally, we identified an association between CC in RR-MS patients and NEDA-3 after three years of follow-up ( p = 0.007). (4) We describe, for the first time, the role of an SNP of the IL-5 gene in regulating central neuroinflammation and influencing clinical course in MS patients.

MiR-142-3p has recently emerged as key factor in tailoring personalized treatments for multiple sclerosis (MS), a chronic autoimmune demyelinating disease of the central nervous system (CNS) with heterogeneous pathophysiology and an unpredictable course. With its involvement in a detrimental regulatory axis with interleukin-1beta (IL1β), miR-142-3p orchestrates excitotoxic synaptic alterations that significantly impact both MS progression and therapeutic outcomes. In this study, we investigated for the first time the influence of individual genetic variability on the miR-142-3p excitotoxic effect in MS. We specifically focused on the single-nucleotide polymorphism Val66Met (rs6265) of the brain-derived neurotrophic factor ( BDNF ) gene, known for its crucial role in CNS functioning. We assessed the levels of miR-142-3p and IL1β in cerebrospinal fluid (CSF) obtained from a cohort of 114 patients with MS upon diagnosis. By stratifying patients according to their genetic background, statistical correlations with clinical parameters were performed. Notably, in Met-carrier patients, we observed a decoupling of miR-142-3p levels from IL1β levels in the CSF, as well as from of disease severity (Expanded Disability Status Score, EDSS; Multiple Sclerosis Severity Score, MSSS; Age-Related Multiple Sclerosis Severity Score, ARMSS) and progression (Progression Index, PI). Our discovery of the interference between BDNF Val66Met polymorphism and the synaptotoxic IL1β-miR-142-3p axis, therefore hampering miR-142-3p action on MS course, provides valuable insights for further development of personalized medicine in the field.

Introduction: Pure hereditary spastic paraplegia (SPG) type 4 (SPG4) is caused by mutations of SPAST gene. This study aimed to analyze SPAST variants in SPG4 patients to highlight the occurrence of splicing mutations and combine functional studies to assess the relevance of these variants in the molecular mechanisms of the disease., Methods: We performed an NGS panel in 105 patients, in silico analysis for splicing mutations, and in vitro minigene assay., Results and Discussion: The NGS panel was applied to screen 105 patients carrying a clinical phenotype corresponding to upper motor neuron syndrome (UMNS), selectively affecting motor control of lower limbs. Pathogenic mutations in SPAST were identified in 12 patients (11.42%), 5 missense, 3 frameshift, and 4 splicing variants. Then, we focused on the patients carrying splicing variants using a combined approach of in silico and in vitro analysis through minigene assay and RNA, if available. For two splicing variants (i.e., c.1245+1G>A and c.1414-2A>T), functional assays confirm the types of molecular alterations suggested by the in silico analysis (loss of exon 9 and exon 12). In contrast, the splicing variant c.1005-1delG differed from what was predicted (skipping exon 7), and the functional study indicates the loss of frame and formation of a premature stop codon. The present study evidenced the high splice variants in SPG4 patients and indicated the relevance of functional assays added to in silico analysis to decipher the pathogenic mechanism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Ferese, Scala, Suppa, Campopiano, Asci, Zampogna, Chiaravalloti, Griguoli, Storto, Pardo, Giardina, Zampatti, Fornai, Novelli, Fanelli, Zecca, Logroscino, Centonze and Gambardella.)

Background and Objective: Early-onset Parkinson's disease (EOPD) commonly recognizes a genetic basis; thus, patients with EOPD are often addressed to diagnostic testing based on next-generation sequencing (NGS) of PD-associated multigene panels. However, NGS interpretation can be challenging in a diagnostic setting, and few studies have addressed this issue so far., Methods: We retrospectively collected data from 648 patients with PD with age at onset younger than 55 years who underwent NGS of a minimal shared panel of 15 PD-related genes, as well as PD-multiplex ligation-dependent probe amplification in eight Italian diagnostic laboratories. Data included a minimal clinical dataset, the complete list of variants included in the diagnostic report, and final interpretation (positive/negative/inconclusive). Patients were further stratified based on age at onset ≤40 years (very EOPD, n = 157). All variants were reclassified according to the latest American College of Medical Genetics and Genomics criteria. For classification purposes, PD-associated GBA1 variants were considered diagnostic., Results: In 186 of 648 (29%) patients, the diagnostic report listed at least one variant, and the outcome was considered diagnostic (positive) in 105 (16%). After reanalysis, diagnosis changed in 18 of 186 (10%) patients, with 5 shifting from inconclusive to positive and 13 former positive being reclassified as inconclusive. A definite diagnosis was eventually reached in 97 (15%) patients, of whom the majority carried GBA1 variants or, less frequently, biallelic PRKN variants. In 89 (14%) cases, the genetic report was inconclusive., Conclusions: This study attempts to harmonize reporting of PD genetic testing across several diagnostic labs and highlights current difficulties in interpreting genetic variants emerging from NGS-multigene panels, with relevant implications for counseling. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., (© 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

Parkinson's disease (PD) is the fastest-growing neurodegenerative disorder, currently affecting ~7 million people worldwide. PD is clinically and genetically heterogeneous, with at least 10% of all cases explained by a monogenic cause or strong genetic risk factor. However, the vast majority of our present data on monogenic PD is based on the investigation of patients of European White ancestry, leaving a large knowledge gap on monogenic PD in underrepresented populations. Gene-targeted therapies are being developed at a fast pace and have started entering clinical trials. In light of these developments, building a global network of centers working on monogenic PD, fostering collaborative research, and establishing a clinical trial-ready cohort is imperative. Based on a systematic review of the English literature on monogenic PD and a successful team science approach, we have built up a network of 59 sites worldwide and have collected information on the availability of data, biomaterials, and facilities. To enable access to this resource and to foster collaboration across centers, as well as between academia and industry, we have developed an interactive map and online tool allowing for a quick overview of available resources, along with an option to filter for specific items of interest. This initiative is currently being merged with the Global Parkinson's Genetics Program (GP2), which will attract additional centers with a focus on underrepresented sites. This growing resource and tool will facilitate collaborative research and impact the development and testing of new therapies for monogenic and potentially for idiopathic PD patients., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Roy N. Alcalay received consulting fees from Avrobio, Caraway, Ono Therapeutics, GSK, Merck, Sanofi, Janssen and grants from the Michael J. Fox Foundation, DOD, the Parkinson’s Disease Foundation, and the NIH. Melinda Barkhuizen received a PhD scholarship from the National Research Foundation of South Africa (grant numbers 89230 and 98217) and internal funding from the research center where the study was conducted (DST/NWU Preclinical Drug Development Platform, North-West University, South Africa); Ampath Pathology laboratories in South Africa donated services in the form of drawing participant blood for DNA extractions, Several neurologists in South Africa assisted the authors with identifying patients with Parkinson`s disease and referring them to the genotyping study; the North-West University, South Africa provided Ethical oversight and approval of the genotyping project. Vincenzo Bonifati received grants from Stichting Parkinson Fonds (Netherlands) and Alzheimer Nederland, he received honoraria from the International Parkinson and Movement Disorder Society (MDS), for lectures and as Chair of the International Congress Scientific Program Committee (2020-2021), and from Elsevier Ltd. as co-Editor in Chief of the journal Parkinsonism & Related Disorders (2018-current); he is unpaid member of the Stichting Parkinson Fonds (Netherlands). Kathrin Brockmann received grants from the German Federal Ministry of Education and Research (BMBF; PDStrat; FKZ 031L0137B) and from the German Center for Neurodegenerative Diseases (DZNE), consulting fees from Abbvie, Lundbeck, UCB, Zambon, Roche, and honoraria from Abbvie and UCB. Jordi Clarimon is full-time employee at Lundbeck A/S (Denmark). Mario Cornejo-Olivas has subcontracts with Cleveland Clinic and San Marcos Foundation for recruiting participants for the LARGE PD study in Peru. Jean-Christophe Corvol received grants from Sanofi and the Michael J. Fox Foundation, consulting fees from Biogen, UCB, Denali, Idorsia, Prevail therapeutics, Theranexus, and honoraria from Biogen. Joana Damásio received honoraria from Zambon Pharmaceuticals. Anna De Rosa received grants for ROPAD – the Rostock International Parkinson’s Disease Study, sponsored by Centogene and grants from Zambon and AIFA (Italian Agency of Drug), and she is member of the advisory board at BIAL. Joaquim Ferreira received grants from Fundação MSD (Portugal), Novartis, Medtronic, and Abbvie, and lecture fees from Lundbeck, Abbvie, BIAL, Biogen, Sunovion Pharmaceuticals, ONO, Affiris, and Zambon, payment for expert testimony from Novartis, and he participates in advisory boards of Lundbeck, Abbvie, BIAL, Affiris, Sunovion Pharmaceuticals, and Zambon; he is employed by CNS (Campus Néurologico Sénior) and the Medical Faculty of Lisbon. Emilia Gatto received consulting fees from Bago Argentina, honoraria from Bago Argentina,UCB, IPMDS, and Europharm, and participates in advisory boards of Bago Argentina and UCB. Nir Giladi received grants from the Michael J Fox Foundation, The National Parkinson Foundation, The European Union, The Israel Science Foundation Teva, NNE program, Biogen, Ionis, Sieratzki Family Foundation, and The Aufzien Academic Center in Tel-Aviv University; he holds royalties or licenses at Lysosomal Therapeutics (LTI); he received consulting fees from Sionara, NeuroDerm, Pharma2B, Denali, Neuron23, Sanofi-Genzyme, Biogen, and Abbvie; he received honoraria from Abbvie, Sanofi-Genzyme, and the Movement Disorder Society. Nobutaka Hattori received grants from the Japan Society for the Promotion of Science (JSPS; 18H04043, 21H04820, 19K22603), the Japan Agency for Medical Research and Development (AMED; JP20dm0307101, JP20dm0207070, JP20ek0109358, JP19ek0109393, JP19gm0710011, JP19km0405206), Health Labour Sciences Research Grant (20FC1049, H29-FC1-062, H29-FC1-033), and the Japan Science and Technology Agency (JPMJMS2024-5); he participates in advisory boards at Dai-Nippon Sumitomo Pharma, Takeda Pharmaceutical, Kyowa Kirin, TEIJIN PHARMA LIMITED, Novartis Pharma, Ono Pharmaceutical, Biogen Idec Japan, and Kissei Pharmaceutical; he receives consulting fees from Hisamitsu Pharma; he received honoraria from Dai-Nippon Sumitomo Pharma, Takeda Pharmaceutical, Kyowa Kirin, AbbVie GK, Nippon Boehringer Ingelheim, Otsuka Pharmaceutical, Novartis Pharma, Bristol-Myers Squibb, Ono Pharmaceutical, FP Pharmaceutical, Eisai, Kissei Pharmaceutical, Nihon Medi-physics, and Daiichi Sankyo; he received payment for expert testimony from Mitsubishi Tanabe Pharma; he received support for attending meeting from Takeda Pharmaceutical, and Kyowa Kirin; he has 17 patents planned or pending and 5 issued; he is Team Leader at the Neurodegenerative Disorders Collaborative Laboratory, RIKEN Center for Brain Science; he holds equity stock (8%) of PARKINSON Laboratories Co. Ltd., unrelated to the submitted work. Silvia Jesús has received grants from “Consejería de Salud y Familias” PI-0459-2018, “acción B Clínicos-Investigdores” B-0007-2019 and from the “instituto de Salud Carlos III” PI-18/01898; she received honoraria from Abbvie, Bial, Merz, UCB, Italfarmaco, Zambon and Server. Valtteri Kaasinen received grants from Turku University Foundation, Päivikki and Sakari Sohlberg Foundation, and the Finnish Cultural Foundation; he received consulting fees from Nordic Infucare, Abbvie, and Adamant Health; he received honoraria from Nordic Infucare and Abbvie; he received Support for attending meetings and/or travel from Nordic Infucare; he participates on a Data Safety Monitoring Board or Advisory Board at Nordic Infucare and Abbvie; he is board member of the Finnish Neurological Society. Christine Klein received royalties from Oxford University Press; she received consulting fees from Centogene and Retromer Therapeutics; she received payment or honoraria from Desitin Pharma; she received support to attend meetings from the Movement Disorder Society; she participates on a Data Safety Monitoring Board or Advisory Board at the Else Kroener Fresenius Foundation. Peter Klivenyi received honoraria from Abbvie and Medtronic. Vladimir Kostic received Grant 17590 from the Ministry of Education, Science and Technological Development of Serbia; he received honoraria from Novartis and Boehringer Ingelheim. Shen-Yang Lim received the following grants, (i) Stipend - Global Parkinson’s Genetics Program (GP2) Working Group Co-Lead Award, from the Michael J. Fox Foundation, (ii) Michael J. Fox Foundation (Grant 18305: "Identifying leucocyte and urine biomarkers in PD patients with LRRK2 G2385R variant"). George Mellick received grants from the National Health and Medical Research Foundation, Australia (APP1151854, APP1084560). Lukasz Milanowski received grants from the Polish National Agency for Academic Exchange Iwanowska’s Fellowship PPN/IWA/2018/1/00006/U/00001/01, the APDA, the Foundation for Polish Science (FNP) and the Haworth Family Professorship in Neurodegenerative Diseases Fund. Pablo mir received grants from the Spanish Ministry of Science and Innovation (RTC2019-007150-1), the Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (ISCIII-FEDER, PI19/01576), and the Consejería de Salud y Bienestar Social de la Junta de Andalucía (PE-0210-2018); he received payment or honoraria for lectures from Abbvie, Abbott, and Zambon; he received Support for attending meetings and travel from Abbvie. Shalini Padmanabhan is employed by the Michael J. Fox Foundation. Andreas Puschmann received grants from Region Skåne, Sweden, Skåne University Hospital, Sweden, The Swedish Parkinson Academy, The Swedish Parkinson Foundation (Parkinsonfonden), MultiPark – a strategic research environment at Lund University, and Bundy Academy, Sweden; he received honoraria from Elsevier (Associate editor for Parkinsonism and Related Disorders), from the International Parkinson and Movement Disorder Society (MDS), and from the International Association of Parkinsonism and Related Disorders; he received support for travels from the International Association of Parkinsonism and Related Disorders; he is board member of the International Association of Parkinsonism and Related Disorders. Mariana Spitz received funding from Centogene for the ROPAD study; she received support for travels from Roche. Justin Solle is employed by the Michel J. Fox Foundation. Oksana Suchowersky received grants from Roche and WaveLifeSciences; she holds royalties or licenses at UpToDate; she received consulting fees from Abbvie and Sunovion; she received honoraria from the World Parkinson Conference; she participates in advisory boards at Alexion; she is board member of the Parkinson Society Alberta. Pichet Termsarasab received book royalties from Elsevier and Springer Nature Switzerland; he received honoraria for manuscript writing from MedLink Neurology and for lectures from Viatris. Bart van de Warrenburg received grants from Radboudumc, ZonMW, Hersenstichting, Gossweiler Foundation, and the Michael J Fox Foundation; he holds royalties or licenses at BSL Springer Nature; he received consulting fees from uniQure; he received honoraria from UKM Medical Center Kuala Lumpur; he participates in Medical advisory board of patient organizations (unpaid); he is unpaid member of Steering committees or executive boards of various research consortia; he received a wearable sensor set by Brugling Fund/Hersenstichting. Caroline H. Williams-Gray collaborates with Astra-Zeneca on the microbiome in Parkinson’s disease; she received consultancy fees from Modus Outcomes and Evidera, Inc./GlaxoSmithKline; she received honoraria from Profile Pharma Limited. Eva-Juliane Vollstedt, Harutyun Madoev, Anna Aasly, Azlina Ahmad-Annuar, Bashayer Al-Mubarak, Victoria Alvarez, Ignacio Amorin, Grazia Annesi, David Arkadir, Soraya Bardien, Roger A. Barker, A. Nazli Basak, Agnita Boon, Laura Brighina, Andrea Carmine Belin, Jonathan Carr, Leonor Correia Guedes, David Crosiers, Parimal Das, Patricia de Carvalho Aguiar, Jolanta Dorszewska, Sibel Ertan, Rosangela Ferese, Gençer Genç, Pilar Gómez-Garre, Hasmet Hanagasi, Faycal Hentati, Dorota Hoffman-Zacharska, Sergey N. Illarioshkin, Joseph Jankovic, Anneke Kievit, Dariusz Koziorowski, Andrea A. Kühn, Anthony E. Lang, Chin-Hsien Lin, Katja Lohmann, Vladana Markovic, Mika Henrik Martikainen, Marcelo Merello, Özgür Öztop-Çakmak, Márcia Mattos Gonçalves Pimentel, Teeratorn Pulkes, Ekaterina Rogaeva, Esther M. Sammler, Maria Skaalum Petersen, Matej Skorvanek, Ai Huey Tan, Avner Thaler, Vitor Tumas, Enza Maria Valente, Ruey-Mei Wu, Baorong Zhang, and Alexander Zimprich report no conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Vollstedt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Individual genetic variability may influence the course of multiple sclerosis (MS). The interleukin (IL)-8C>T rs2227306 single nucleotide polymorphism (SNP) regulates IL-8 activity in other clinical conditions; however, its role in MS has never been investigated., Objectives: To explore the association between IL-8 SNP rs2227306, cerebrospinal fluid (CSF) IL-8 concentrations, clinical, and radiological characteristics in a group of newly diagnosed MS patients., Methods: In 141 relapsing-remitting (RR)-MS patients, rs2227306 polymorphism, CSF levels of IL-8, clinical, and demographical characteristics were determined. In 50 patients, structural magnetic resonance imaging (MRI) measures were also assessed., Results: An association between CSF IL-8 and Expanded Disability Status Scale (EDSS) at diagnosis was found in our set of patients ( r  = 0.207, p  = 0.014). CSF IL-8 concentrations were significantly higher in patients carrying the T variant of rs2227306 ( p  = 0.004). In the same group, a positive correlation emerged between IL-8 and EDSS ( r  = 0.273, p  = 0.019). Finally, a negative correlation between CSF levels of IL-8 and cortical thickness emerged in rs2227306T carriers ( r  = -0.498, p  = 0.005)., Conclusion: We describe for the first time a role of SNP rs2227306 of IL-8 gene in regulating the expression and the activity of this inflammatory cytokine in MS.

Background: As gene-targeted therapies are increasingly being developed for Parkinson's disease (PD), identifying and characterizing carriers of specific genetic pathogenic variants is imperative. Only a small fraction of the estimated number of subjects with monogenic PD worldwide are currently represented in the literature and availability of clinical data and clinical trial-ready cohorts is limited., Objective: The objectives are to (1) establish an international cohort of affected and unaffected individuals with PD-linked variants; (2) provide harmonized and quality-controlled clinical characterization data for each included individual; and (3) further promote collaboration of researchers in the field of monogenic PD., Methods: We conducted a worldwide, systematic online survey to collect individual-level data on individuals with PD-linked variants in SNCA, LRRK2, VPS35, PRKN, PINK1, DJ-1, as well as selected pathogenic and risk variants in GBA and corresponding demographic, clinical, and genetic data. All registered cases underwent thorough quality checks, and pathogenicity scoring of the variants and genotype-phenotype relationships were analyzed., Results: We collected 3888 variant carriers for our analyses, reported by 92 centers (42 countries) worldwide. Of the included individuals, 3185 had a diagnosis of PD (ie, 1306 LRRK2, 115 SNCA, 23 VPS35, 429 PRKN, 75 PINK1, 13 DJ-1, and 1224 GBA) and 703 were unaffected (ie, 328 LRRK2, 32 SNCA, 3 VPS35, 1 PRKN, 1 PINK1, and 338 GBA). In total, we identified 269 different pathogenic variants; 1322 individuals in our cohort (34%) were indicated as not previously published., Conclusions: Within the MJFF Global Genetic PD Study Group, we (1) established the largest international cohort of affected and unaffected individuals carrying PD-linked variants; (2) provide harmonized and quality-controlled clinical and genetic data for each included individual; (3) promote collaboration in the field of genetic PD with a view toward clinical and genetic stratification of patients for gene-targeted clinical trials. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society., (© 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.)

Cells from glioblastoma multiforme (GBM) feature up-regulation of the mechanistic Target of Rapamycin (mTOR), which brings deleterious effects on malignancy and disease course. At the cellular level, up-regulation of mTOR affects a number of downstream pathways and suppresses autophagy, which is relevant for the neurobiology of GBM. In fact, autophagy acts on several targets, such as protein clearance and mitochondrial status, which are key in promoting the malignancy GBM. A defective protein clearance extends to cellular prion protein (PrPc). Recent evidence indicates that PrPc promotes stemness and alters mitochondrial turnover. Therefore, the present study measures whether in GBM cells abnormal amount of PrPc and mitochondrial alterations are concomitant in baseline conditions and whether they are reverted by mTOR inhibition. Proteins related to mitochondrial turnover were concomitantly assessed. High amounts of PrPc and altered mitochondria were both mitigated dose-dependently by the mTOR inhibitor rapamycin, which produced a persistent activation of the autophagy flux and shifted proliferating cells from S to G1 cell cycle phase. Similarly, mTOR suppression produces a long-lasting increase of proteins promoting mitochondrial turnover, including Pink1/Parkin. These findings provide novel evidence about the role of autophagy in the neurobiology of GBM.

Triplication of the SNCA gene, encoding the protein alpha-Synuclein (αSyn), is a rare cause of aggressive and early-onset parkinsonism. Herein, we generated iPSCs from two siblings with a recently described compact SNCA gene triplication and suffering from severe motor impairments, psychiatric symptoms, and cognitive deterioration. Using CRISPR/Cas9 gene editing, each SNCA copy was inactivated by targeted indel mutations generating a panel of isogenic iPSCs with a decremental number from 4 down to none of functional SNCA gene alleles. We differentiated these iPSC lines in midbrain dopaminergic (DA) neuronal cultures to characterize αSyn aggregation in native and seeded conditions and evaluate its associated cellular dysfunctions. Utilizing a new nanobody-based biosensor combined with super-resolved imaging, we were able to visualize and measure αSyn aggregates in early DA neurons in unstimulated conditions. Calcium dysregulation and mitochondrial alterations were the first pathological signs detectable in early differentiated DA neuronal cultures. Accelerated αSyn aggregation was induced by exposing neurons to structurally well-characterized synthetic αSyn fibrils. 4xSNCA DA neurons showed the highest vulnerability, which was associated with high levels of oxidized DA and amplified by TAX1BP1 gene disruption. Seeded DA neurons developed large αSyn deposits whose morphology and internal constituents resembled Lewy bodies commonly observed in Parkinson's disease (PD) patient brain tissues. These findings provide strong evidence that this isogenic panel of iPSCs with SNCA multiplications offers a remarkable cellular platform to investigate mechanisms of PD and validate candidate inhibitors of native and seeded αSyn aggregation., (© 2022. The Author(s).)

(1) Background: The clinical course of multiple sclerosis (MS) is critically influenced by the expression of different pro-inflammatory and anti-inflammatory cytokines. Interleukin 6 (IL-6) represents a major inflammatory molecule previously associated with exacerbated disease activity in relapsing remitting MS (RR-MS); however, the role of single-nucleotide polymorphisms (SNPs) in the IL-6 gene has not been fully elucidated in MS. (2) Methods: We explored in a cohort of 171 RR-MS patients, at the time of diagnosis, the associations between four IL-6 SNPs ( rs1818879 , rs1554606, rs1800797 , and rs1474347 ), CSF inflammation, and clinical presentation. (3) Results: Using principal component analysis and logistic regression analysis we identified an association between rs1818879 , radiological activity, and a set of cytokines, including the IL-1 β , IL-9, IL-10, and IL-13. No significant associations were found between other SNPs and clinical or inflammatory parameters. (4) Conclusions: The association between the rs1818879 polymorphism and subclinical neuroinflammatory activity suggests that interindividual differences in the IL-6 gene might influence the immune activation profile in MS.

Alpha-synuclein (α-syn) is a protein considered to be detrimental in a number of degenerative disorders (synucleinopathies) of which α-syn aggregates are considered a pathological hallmark. The clearance of α-syn strongly depends on autophagy, which can be stimulated by inhibiting the mechanistic target of rapamycin (mTOR). Thus, the overexpression of mTOR and severe autophagy suppression may produce α-syn accumulation, including the proteinase K-resistant protein isoform. Glioblastoma multiforme (GBM) is a lethal brain tumor that features mTOR overexpression and severe autophagy inhibition. Cell pathology in GBM is reminiscent of a fast, progressive degenerative disorder. Therefore, the present work questions whether, as is analogous to neurons during degenerative disorders, an overexpression of α-syn occurs within GBM cells. A high amount of α-syn was documented in GBM cells via real-time PCR (RT-PCR), Western blotting, immunohistochemistry, immuno-fluorescence, and ultrastructural stoichiometry, compared with the amount of β- and γ-synucleins and compared with the amount of α-syn counted within astrocytes. The present study indicates that (i) α-syn is overexpressed in GBM cells, (ii) α-syn expression includes a proteinase-K resistant isoform, (iii) α-syn is dispersed from autophagy-like vacuoles to the cytosol, (iv) α-syn overexpression and cytosol dispersion are mitigated by rapamycin, and (v) the α-syn-related GBM-like phenotype is mitigated by silencing the SNCA gene.

The clinical course of multiple sclerosis (MS) is critically influenced by the interplay between inflammatory and neurodegenerative processes. The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism (rs6265), one of the most studied single-nucleotide polymorphisms (SNPs), influences brain functioning and neurodegenerative processes in healthy individuals and in several neuropsychiatric diseases. However, the role of this polymorphism in MS is still controversial. In 218 relapsing-remitting (RR)-MS patients, we explored, at the time of diagnosis, the associations between the Val66Met polymorphism, clinical characteristics, and the cerebrospinal fluid (CSF) levels of a large set of pro-inflammatory and anti-inflammatory molecules. In addition, associations between Val66Met and structural MRI measures were assessed. We identified an association between the presence of Met and a combination of cytokines, identified by principal component analysis (PCA), including the pro-inflammatory molecules MCP-1, IL-8, TNF, Eotaxin, and MIP-1b. No significant associations emerged with clinical characteristics. Analysis of MRI measures evidenced reduced cortical thickness at the time of diagnosis in patients with Val66Met. We report for the first time an association between the Val66Met polymorphism and central inflammation in MS patients at the time of diagnosis. The role of this polymorphism in both inflammatory and neurodegenerative processes may explain its complex influence on the MS course.

Peripheral markers in Parkinson's disease (PD) represent a hot issue to provide early diagnosis and assess disease progression. The gold standard marker of PD should feature the same reliability as the pathogenic alteration, which produces the disease itself. PD is foremost a movement disorder produced by a loss of nigrostriatal dopamine innervation, in which striatal dopamine terminals are always markedly reduced in PD patients to an extent, which never overlaps with controls. Similarly, a reliable marker of PD should possess such a non-overlapping feature when compared with controls. In the present study, we provide a novel pathological hallmark, the autophagosome, which in each PD patient was always suppressed compared with each control subject. Autophagosomes were counted as microtubule-associated proteins 1A/1B light chain 3B (LC3)-positive vacuoles at ultrastructural morphometry within peripheral (blood) blood mononuclear cells (PBMC). This also provides the gold standard to assess the autophagy status. Since autophagy may play a role in the pathogenesis of PD, autophagosomes may be a disease marker, while participating in the biology of the disease. Stoichiometric measurement of α-synuclein despite significantly increased in PD patients, overlapped between PD and control patients. Although the study need to be validated in large populations, the number of autophagy vacuoles is neither related with therapy (the amount was similarly suppressed in a few de novo patients), nor the age in PD or controls., (© 2021. The Author(s).)

The peri-infarct region, which surrounds the irreversible ischemic stroke area is named ischemic penumbra . This term emphasizes the borderline conditions for neurons placed within such a critical region. Area penumbra separates the ischemic core, where frank cell loss occurs, from the surrounding healthy brain tissue. Within such a brain region, nervous matter, and mostly neurons are impaired concerning metabolic conditions. The classic biochemical marker, which reliably marks area penumbra is the over-expression of the heat shock protein 70 (HSP70). However, other proteins related to cell clearing pathways are modified within area penumbra . Among these, autophagy proteins like LC3 increase in a way, which recapitulates Hsp70. In contrast, components, such as P20S, markedly decrease. Despite apparent discrepancies, the present study indicates remarkable overlapping between LC3 and P20S redistribution within area penumbra . In fact, the amount of both proteins is markedly reduced within vacuoles. Specifically, a massive loss of LC3 + P20S immuno-positive vacuoles (autophagoproteasomes) is reported here. This represents the most relevant sub-cellular alteration here described in cell clearing pathways within area penumbra . The functional significance of these findings remains to be determined and it will take a novel experimental stream to decipher the fine-tuning of such a phenomenon.

Charcot-Marie-Tooth (CMT) disease is the most prevalent inherited motor sensory neuropathy, which clusters a clinically and genetically heterogeneous group of disorders with more than 90 genes associated with different phenotypes. The goal of this study is to identify the genetic features in the recruited cohort of patients, highlighting the role of rare variants in the genotype-phenotype correlation. We enrolled 67 patients and applied a diagnostic protocol including multiple ligation-dependent probe amplification for copy number variation (CNV) detection of PMP22 locus, and next-generation sequencing (NGS) for sequencing of 47 genes known to be associated with CMT and routinely screened in medical genetics. This approach allowed the identification of 26 patients carrying a whole gene CNV of PMP22 . In the remaining 41 patients, NGS identified the causative variants in eight patients in the genes HSPB1 , MFN2 , KIF1A , GDAP1 , MTMR2 , SH3TC2 , KIF5A , and MPZ (five new vs. three previously reported variants; three sporadic vs. five familial variants). Familial segregation analysis allowed to correctly interpret two variants, initially reported as "variants of uncertain significance" but re-classified as pathological. In this cohort is reported a patient carrying a novel familial mutation in the tail domain of KIF5A [a protein domain previously associated with familial amyotrophic lateral sclerosis (ALS)], and a CMT patient carrying a HSPB1 mutation, previously reported in ALS. These data indicate that combined tools for gene association in medical genetics allow dissecting unexpected phenotypes associated with previously known or unknown genotypes, thus broadening the phenotype expression produced by either pathogenic or undefined variants. Clinical trial registration : ClinicalTrials.gov (NCT03084224)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ferese, Campopiano, Scala, D’Alessio, Storto, Buttari, Centonze, Logroscino, Zecca, Zampatti, Fornai, Cianci, Manfroi, Giardina, Magnani, Suppa, Novelli and Gambardella.)

Glioblastoma (GBM) cells feature mitochondrial alterations, which are documented and quantified in the present study, by using ultrastructural morphometry. Mitochondrial impairment, which roughly occurs in half of the organelles, is shown to be related to mTOR overexpression and autophagy suppression. The novelty of the present study consists of detailing an mTOR-dependent mitophagy occlusion, along with suppression of mitochondrial fission. These phenomena contribute to explain the increase in altered mitochondria reported here. Administration of the mTOR inhibitor rapamycin rescues mitochondrial alterations. In detail, rapamycin induces the expression of genes promoting mitophagy ( PINK1 , PARKIN , ULK1 , AMBRA1 ) and mitochondrial fission ( FIS1 , DRP1 ). This occurs along with over-expression of VPS34 , an early gene placed upstream in the autophagy pathway. The topographic stoichiometry of proteins coded by these genes within mitochondria indicates that, a remarkable polarization of proteins involved in fission and mitophagy within mitochondria including LC3 takes place. Co-localization of these proteins within mitochondria, persists for weeks following rapamycin, which produces long-lasting mitochondrial plasticity. Thus, rapamycin restores mitochondrial status in GBM cells. These findings add novel evidence about mitochondria and GBM, while fostering a novel therapeutic approach to restore healthy mitochondria through mTOR inhibition.

X-linked adrenoleukodystrophy (X-ALD, OMIM #300100) is the most common peroxisomal disorder clinically characterized by two main phenotypes: adrenomyeloneuropathy (AMN) and the cerebral demyelinating form of X-ALD (cerebral ALD). The disease is caused by defects in the gene for the adenosine triphosphate (ATP)-binding cassette protein, subfamily D ( ABCD1 ) that encodes the peroxisomal transporter of very-long-chain fatty acids (VLCFAs). The defective function of ABCD1 protein prevents β-oxidation of VLCFAs, which thus accumulate in tissues and plasma, to represent the hallmark of the disease. As in many X-linked diseases, it has been routinely expected that female carriers are asymptomatic. Nonetheless, recent findings indicate that most ABCD1 female carriers become symptomatic, with a motor disability that typically appears between the fourth and fifth decade. In this paper, we report a large family in which affected males died during the first decade, while affected females develop, during the fourth decade, progressive lower limb weakness with spastic or ataxic-spastic gait, tetra-hyperreflexia with sensory alterations. Clinical and genetic evaluations were performed in nine subjects, eight females (five affected and three healthy) and one healthy male. All affected females were carriers of the c.1661G>A (p.Arg554His, rs201568579) mutation. This study strengthens the relevance of clinical symptoms in female carriers of ABCD1 mutations, which leads to a better understanding of the role of the genetic background and the genotype-phenotype correlation. This indicates the relevance to include ABCD1 genes in genetic panels for gait disturbance in women.

Background: Levodopa (L-Dopa), representing the therapeutic gold standard for the treatment of Parkinson disease (PD), is associated with side effects like L-Dopa induced dyskinesia (LID). Although several non-genetic and genetic factors have been investigated for association with LID risk, contrasting results were reported and its genetic basis remain largely unexplored. Methods: In an Italian PD cohort (N = 460), we first performed stepwise multivariable Cox Proportional Hazard regressions modeling LID risk as a function of gender, PD familiarity, clinical subtype, weight, age-at-onset (AAO) and years-of-disease (YOD), L-Dopa dosage, severity scores, and scales assessing motor (UPDRS-III), cognitive (MoCA), and non-motor symptoms (NMS). Then we enriched the resulting model testing two variants-rs356219 and D4S3481-increasing the expression of the SNCA gene, previously suggested as a potential mechanism of LID onset. To account for more complex (non-linear) relations of these variables with LID risk, we built a survival random forest (SRF) algorithm including all the covariates mentioned above. Results: Among tested variables (N = 460 case-complete, 211 LID events; total follow-up 31,361 person-months, median 61 months), disease duration showed significant association ( p < 0.005), with 6 (3-8)% decrease of LID risk per additional YOD. Other nominally significant associations were observed for gender-with women showing a 39 (5-82)% higher risk of LID-and AAO, with 2 (0.3-3)% decrease of risk for each year increase of PD onset. The SRF algorithm confirmed YOD as the most prominent feature influencing LID risk, with a variable importance of about 8% in the model. In genetic models, no statistically significant effects on incident LID risk was observed. Conclusions: This evidence supports a protective effect of late PD onset and gender (men) against LID risk and suggests a new independent protective factor, YOD. Moreover, it underlines the importance of personalized therapeutic protocols for PD patients in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tirozzi, Modugno, Palomba, Ferese, Lombardi, Olivola, Gialluisi and Esposito.)

The neurotoxin 1-methyl, 4-phenyl, 1, 2, 3, 6-tetrahydropiridine (MPTP) is widely used to produce experimental parkinsonism. Such a disease is characterized by neuronal damage in multiple regions beyond the nigrostriatal pathway including the spinal cord. The neurotoxin MPTP damages spinal motor neurons. So far, in Parkinson's disease (PD) patients alpha-synuclein aggregates are described in the dorsal horn of the spinal cord. Nonetheless, no experimental investigation was carried out to document whether MPTP affects the sensory compartment of the spinal cord. Thus, in the present study, we investigated whether chronic exposure to small doses of MPTP (5 mg/kg/X2, daily, for 21 days) produces any pathological effect within dorsal spinal cord. This mild neurotoxic protocol produces a damage only to nigrostriatal dopamine (DA) axon terminals with no decrease in DA nigral neurons assessed by quantitative stereology. In these experimental conditions we documented a decrease in enkephalin-, calretinin-, calbindin D28K-, and parvalbumin-positive neurons within lamina I and II and the outer lamina III. Met-Enkephalin and substance P positive fibers are reduced in laminae I and II of chronically MPTP-treated mice. In contrast, as reported in PD patients, alpha-synuclein is markedly increased within spared neurons and fibers of lamina I and II after MPTP exposure. This is the first evidence that experimental parkinsonism produces the loss of specific neurons of the dorsal spinal cord, which are likely to be involved in sensory transmission and in pain modulation providing an experimental correlate for sensory and pain alterations in PD.

In multiple sclerosis (MS), activated T and B lymphocytes and microglial cells release various proinflammatory cytokines, promoting neuroinflammation and negatively affecting the course of the disease. The immune response homeostasis is crucially regulated by the activity of the enzyme adenosine deaminase (ADA), as evidenced in patients with genetic ADA deficiency and in those treated with cladribine tablets. We investigated in a group of patients with MS the associations of a single nucleotide polymorphism (SNP) of ADA gene with disease characteristics and cerebrospinal fluid (CSF) inflammation. The SNP rs244072 of the ADA gene was determined in 561 patients with MS. Disease characteristics were assessed at the time of diagnosis; furthermore, in 258 patients, proinflammatory and anti-inflammatory molecules were measured in the CSF. We found a significant association between rs244072 and both clinical characteristics and central inflammation. In C-carriers, significantly enhanced disability and increased CSF levels of TNF, IL-5 and RANTES was observed. In addition, lower CSF levels of the anti-inflammatory cytokine IL-10 were found. Finally, the presence of the C allele was associated with a tendency of increased lymphocyte count. In MS patients, ADA SNP rs244072 is associated with CSF inflammation and disability. The selective targeting of the ADA pathway through cladribine tablet therapy could be effective in MS by acting on a pathogenically relevant biological mechanism.

Seizures originating from limbic structures, especially when prolonged for several minutes/hours up to status epilepticus (SE), can cause specific neurodegenerative phenomena in limbic and subcortical structures. The cholinergic nuclei belonging to the basal forebrain (BF) (namely, medial septal nucleus (MSN), diagonal band of Broca (DBB), and nucleus basalis of Meynert (NBM)) belong to the limbic system, while playing a pivotal role in cognition and sleep-waking cycle. Given the strong interconnections linking these limbic nuclei with limbic cortical structures, a persistent effect of SE originating from limbic structures on cBF morphology is plausible. Nonetheless, only a few experimental studies have addressed this issue. In this review, we describe available data and discuss their significance in the scenario of seizure-induced brain damage. In detail, the manuscript moves from a recent study in a model of focally induced limbic SE, in which the pure effects of seizure spreading through the natural anatomical pathways towards the cholinergic nuclei of BF were tracked by neuronal degeneration. In this experimental setting, a loss of cholinergic neurons was measured in all BF nuclei, to various extents depending on the specific nucleus. These findings are discussed in the light of the effects on the very same nuclei following SE induced by systemic injections of kainate or pilocarpine. The various effects including discrepancies among different studies are discussed. Potential implications for human diseases are included.

Background: Leukodystrophies are familial heterogeneous disorders primarily affecting the white matter, which are defined as hypomyelinating or demyelinating based on disease severity as assessed at MRI. Recently, a group of clinically overlapping hypomyelinating leukodystrophies (HL) has been associated with mutations in RNA polymerase III enzymes (Pol III) subunits., Case Presentation: In this manuscript, we describe two Italian siblings carrying a novel POLR3A genotype. MRI imaging, genetic analysis, and clinical data led to diagnosing HL type 7. The female sibling, at the age of 34, is tetra-paretic and suffers from severe cognitive regression. She had a disease onset at the age of 19, characterized by slow and progressive cognitive impairment associated with gait disturbances and amenorrhea. The male sibling was diagnosed during an MRI carried out for cephalalgia at the age of 41. After 5 years, he developed mild cognitive impairment, dystonia with 4-limb hypotonia, and moderate dysmetria with balance and gait impairment., Conclusions: The present study provides the first evidence of unusually late age of onset in HL, describing two siblings with a novel POLR3A genotype which showed the first symptoms at the age of 41 and 19, respectively. This provides a powerful insight into clinical heterogeneity and genotype-phenotype correlation in POLR3A related HL.

In glioblastoma (GBM) cells, an impairment of mitochondrial activity along with autophagy suppression occurs. Autophagy suppression in GBM promotes stemness, invasion, and poor prognosis. The autophagy deficit seems to be due, at least in part, to an abnormal up-regulation of the mammalian target of rapamycin (mTOR), which may be counteracted by pharmacological mTORC1 inhibition. Since autophagy activation is tightly bound to increased mitochondriogenesis, a defect in the synthesis of novel mitochondria is expected to occur in GBM cells. In an effort to measure a baseline deficit in mitochondria and promote mitochondriogenesis, the present study used two different GBM cell lines, both featuring mTOR hyperactivity. mTORC1 inhibition increases the expression of genes and proteins related to autophagy, mitophagy, and mitochondriogenesis. Autophagy activation was counted by RT-PCR of autophagy genes, LC3-&nbsp;immune-fluorescent puncta and immune-gold, as well as specific mitophagy-dependent BNIP3 stoichiometric increase in situ, within mitochondria. The activation of autophagy-related molecules and organelles after rapamycin exposure occurs concomitantly with progression of autophagosomes towards lysosomes. Remarkably, mitochondrial biogenesis and plasticity (increased mitochondrial number, integrity, and density as well as decreased mitochondrial area) was long-&nbsp;lasting for weeks following rapamycin withdrawal., Competing Interests: The authors declare no conflict of interest.

Recently, several studies focused on the genetics of gliomas. This allowed identifying several germline loci that contribute to individual risk for tumor development, as well as various somatic mutations that are key for disease classification. Unfortunately, none of the germline loci clearly confers increased risk per se. Contrariwise, somatic mutations identified within the glioma tissue define tumor genotype, thus representing valid diagnostic and prognostic markers. Thus, genetic features can be used in glioma classification and guided therapy. Such copious genomic variabilities are screened routinely in glioma diagnosis. In detail, Sanger sequencing or pyrosequencing, fluorescence in-situ hybridization, and microsatellite analyses were added to immunohistochemistry as diagnostic markers. Recently, Next Generation Sequencing was set-up as an all-in-one diagnostic tool aimed at detecting both DNA copy number variations and mutations in gliomas. This approach is widely used also to detect circulating tumor DNA within cerebrospinal fluid from patients affected by primary brain tumors. Such an approach is providing an alternative cost-effective strategy to genotype all gliomas, which allows avoiding surgical tissue collection and repeated tumor biopsies. This review summarizes available molecular features that represent solid tools for the genetic diagnosis of gliomas at present or in the next future.

Methamphetamine (Meth) produces a variety of epigenetic effects in the brain, which are seminal to establish long-lasting alterations in neuronal activity. However, most epigenetic changes were described by measuring the rough amount of either histone acetylation and methylation or direct DNA methylation, without focusing on a specific DNA sequence. This point is key to comprehend Meth-induced phenotypic changes, brain plasticity, addiction and neurodegeneration. In this research paper we analyze the persistence of Meth-induced striatal synucleinopathy at a prolonged time interval of Meth withdrawal. At the same time, Meth-induced alterations, specifically within alpha-synuclein gene (SNCA) or its promoter, were evaluated. We found that exposure to high and/or prolonged doses of Meth, apart from producing nigro-striatal toxicity, determines a long-lasting increase in striatal alpha-synuclein levels. This is consistent along immune-blotting, immune-histochemistry, and electron microscopy. This was neither associated with an increase of SNCA copy number nor with alterations within SNCA sequence. However, we documented persistently demethylation within SNCA promoter, which matches the increase in alpha-synuclein protein. The amount of the native protein, which was measured stoichiometrically within striatal neurons, surpasses the increase reported following SNCA multiplications. Demethylation was remarkable (ten-fold of controls) and steady, even at prolonged time intervals being tested so far (up to 21 days of Meth withdrawal). Similarly alpha-synuclein protein assayed stoichiometrically steadily increased roughly ten-fold of controls. Meth-induced increase of alpha-synuclein was also described within limbic areas. These findings are discussed in the light of Meth-induced epigenetic changes, Meth-induced phenotype alterations, and Meth-induced neurodegeneration., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)