Maria Teresa Bassi

Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder characterized by severe psychomotor retardation and ophthalmologic abnormalities, including corneal opacity, retinal degeneration, and strabismus. Unlike the situation in other lysosomal disorders, the accumulation of heterogeneous storage material observed in MLIV does not result from a block in the catabolic pathways but is due to an ill-defined transport defect in the late steps of endocytosis. With the aim of cloning the MLIV gene, we searched in the 19p13.2-13.3 region, where the locus previously had been assigned by linkage mapping. In this region, we have identified a novel gene that is mutated in all patients with MLIV who were enrolled in our study. One patient was homozygous for the splice-acceptor mutation, and another was homozygous for a deletion removing the first six exons of the gene. In addition, four compound heterozygotes for these two mutations were identified. Haplotype analysis indicates that we have identified the two major founder mutations, which account for >95% of MLIV chromosomes in Ashkenazi Jewish patients. The gene, <i>ML4,</i> encodes a protein named "mucolipidin," which localizes on the plasma membrane and, in the carboxy-terminal region, shows homologies to polycystin-2, the product of the polycystic kidney disease 2 gene (<i>PKD2</i>) and to the family of transient receptor potential Ca²⁺ channels. Mucolipidin is likely to play an important role in endocytosis.

A large number of genes encoding for tubulin proteins are expressed in the developing brain. Each is subject to specific spatial and temporal expression patterns. However, most are highly expressed in post-mitotic neurons during stages of neuronal migration and differentiation. The major tubulin subclasses (alpha- and beta-tubulin) share high sequence and structural homology. These globular proteins form heterodimers and subsequently co-assemble into microtubules. Microtubules are dynamic, cytoskeletal polymers which play key roles in cellular processes crucial for cortical development, including neuronal proliferation, migration and cortical laminar organisation. Mutations in seven genes encoding alpha-tubulin (TUBA1A), beta-tubulin (TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBB) and gamma-tubulin (TUBG1) isoforms have been associated with a wide and overlapping range of brain malformations or “Tubulinopathies”. The majority of cortical phenotypes include lissencephaly, polymicrogyria, microlissencephaly and simplified gyration. Well-known hallmarks of the tubulinopathies include dysmorphism of the basal ganglia (fusion of the caudate nucleus and putamen with absence of the anterior limb of the internal capsule), midline commissural structures hypoplasia and/or agenesis (anterior commissure, corpus callosum and fornix), hypoplasia of the oculomotor and optic nerves, cerebellar hypoplasia or dysplasia and dysmorphism of the hind-brain structures. The cortical and extra-cortical brain phenotypes observed are largely dependent on the specific tubulin gene affected. In the present review, all the published data on tubulin family gene mutations and the associated cortical phenotypes are summarized. In addition, the most typical neuroimaging patterns of malformations of cortical development associated with tubulin gene mutations detected on the basis of our own experience are described.

Spastic paraplegia type 5 (SPG5) is a rare subtype of hereditary spastic paraplegia, a highly heterogeneous group of neurodegenerative disorders defined by progressive neurodegeneration of the corticospinal tract motor neurons. SPG5 is caused by recessive mutations in the gene CYP7B1 encoding oxysterol-7α-hydroxylase. This enzyme is involved in the degradation of cholesterol into primary bile acids. CYP7B1 deficiency has been shown to lead to accumulation of neurotoxic oxysterols. In this multicentre study, we have performed detailed clinical and biochemical analysis in 34 genetically confirmed SPG5 cases from 28 families, studied dose-dependent neurotoxicity of oxysterols in human cortical neurons and performed a randomized placebo-controlled double blind interventional trial targeting oxysterol accumulation in serum of SPG5 patients. Clinically, SPG5 manifested in childhood or adolescence (median 13 years). Gait ataxia was a common feature. SPG5 patients lost the ability to walk independently after a median disease duration of 23 years and became wheelchair dependent after a median 33 years. The overall cross-sectional progression rate of 0.56 points on the Spastic Paraplegia Rating Scale per year was slightly lower than the longitudinal progression rate of 0.80 points per year. Biochemically, marked accumulation of CYP7B1 substrates including 27-hydroxycholesterol was confirmed in serum (n = 19) and cerebrospinal fluid (n = 17) of SPG5 patients. Moreover, 27-hydroxycholesterol levels in serum correlated with disease severity and disease duration. Oxysterols were found to impair metabolic activity and viability of human cortical neurons at concentrations found in SPG5 patients, indicating that elevated levels of oxysterols might be key pathogenic factors in SPG5. We thus performed a randomized placebo-controlled trial (EudraCT 2015-000978-35) with atorvastatin 40 mg/day for 9 weeks in 14 SPG5 patients with 27-hydroxycholesterol levels in serum as the primary outcome measure. Atorvastatin, but not placebo, reduced serum 27-hydroxycholesterol from 853 ng/ml [interquartile range (IQR) 683–1113] to 641 (IQR 507–694) (−31.5%, P = 0.001, Mann-Whitney U-test). Similarly, 25-hydroxycholesterol levels in serum were reduced. In cerebrospinal fluid 27-hydroxycholesterol was reduced by 8.4% but this did not significantly differ from placebo. As expected, no effects were seen on clinical outcome parameters in this short-term trial. In this study, we define the mutational and phenotypic spectrum of SPG5, examine the correlation of disease severity and progression with oxysterol concentrations, and demonstrate in a randomized controlled trial that atorvastatin treatment can effectively lower 27-hydroxycholesterol levels in serum of SPG5 patients. We thus demonstrate the first causal treatment strategy in hereditary spastic paraplegia.

Tumor microenvironment is fundamental for cancer progression and chemoresistance. Among stromal cells tumor-associated macrophages (TAMs) represent the largest population of infiltrating inflammatory cells in malignant tumors, promoting their growth, invasion and immune evasion. M2-polarized TAMs are endowed with the NO generating enzyme iNOS. NO has divergent effects on tumors, since it can either stimulate tumor cells growth or promote their death depending on the source of it; likewise the role of iNOS in cancer differs depending on the cell type. The role of NO generated by TAM has not been investigated. Using different tumor models in vitro and in vivo we found that nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) of M2-polarized TAMs is able to protect tumor cells from apoptosis induced by the chemotherapeutic agent cisplatin (CDDP). Here, we demonstrate that the protective effect of NO depends on the inhibition of acid sphingomyelinase (A-SMase), which is activated by CDDP in a pathway involving the death receptor CD95. Mechanistic insights indicate that NO actions occur via generation of cyclic GMP and activation of protein kinase G (PKG), inducing phosphorylation of syntaxin 4 (synt4), a SNARE protein responsible for A-SMase trafficking and activation. Noteworthy, phosphorylation of synt4 at serine 78 by PKG is responsible for the proteasome-dependent degradation of synt4, which limits the CDDP-induced exposure of A-SMase to the plasma membrane of tumor cells. This inhibits the cytotoxic mechanism of CDDP reducing A-SMase-triggered apoptosis. This is the first demonstration that endogenous NO system is a key mechanism through which TAMs protect tumor cells from chemotherapeutic drug-induced apoptosis. The identification of the pathway responsible of A-SMase activity downregulation in tumors leading to chemoresistance warrants further investigations as a means to identify new anti-cancer molecules capable to specifically inhibit synt4 degradation.

The endoplasmic reticulum enzyme fatty acid 2-hydroxylase (FA2H) plays a major role in the formation of 2-hydroxy glycosphingolipids, main components of myelin. FA2H deficiency in mice leads to severe central demyelination and axon loss. In humans it has been associated with phenotypes from the neurodegeneration with brain iron accumulation (fatty acid hydroxylase-associated neurodegeneration, FAHN), hereditary spastic paraplegia (HSP type SPG35) and leukodystrophy (leukodystrophy with spasticity and dystonia) spectrum. We performed an in-depth clinical and retrospective neurophysiological and imaging study in a cohort of 19 cases with biallelic FA2H mutations. FAHN/SPG35 manifests with early childhood onset predominantly lower limb spastic tetraparesis and truncal instability, dysarthria, dysphagia, cerebellar ataxia, and cognitive deficits, often accompanied by exotropia and movement disorders. The disease is rapidly progressive with loss of ambulation after a median of 7 years after disease onset and demonstrates little interindividual variability. The hair of FAHN/SPG35 patients shows a bristle-like appearance; scanning electron microscopy of patient hair shafts reveals deformities (longitudinal grooves) as well as plaque-like adhesions to the hair, likely caused by an abnormal sebum composition also described in a mouse model of FA2H deficiency. Characteristic imaging features of FAHN/SPG35 can be summarized by the ‘WHAT’ acronym: white matter changes, hypointensity of the globus pallidus, ponto-cerebellar atrophy, and thin corpus callosum. At least three of four imaging features are present in 85% of FA2H mutation carriers. Here, we report the first systematic, large cohort study in FAHN/SPG35 and determine the phenotypic spectrum, define the disease course and identify clinical and imaging biomarkers.

X-linked heterotaxy (HTX1) is a rare developmental disorder characterized by disturbances in embryonic laterality and other midline developmental field defects. HTX1 results from mutations in ZIC3, a member of the GLI transcription factor superfamily. A targeted deletion of the murine Zic3 locus has been created to investigate its function and interactions with other molecular components of the left-right axis pathway. Embryonic lethality is seen in approximately 50% of null mice with an additional 30% lethality in the perinatal period. Null embryos have defects in turning, cardiac development and neural tube closure. Malformations in live born null mice include complex congenital heart defects, pulmonary reversal or isomerism, CNS defects and vertebral/rib anomalies. Investigation of nodal expression in Zic3-deficient mice indicates that, although nodal is initially expressed symmetrically in the node, there is failure to maintain expression and to shift to asymmetric expression. Subsequent nodal and Pitx2 expression in the lateral plate mesoderm in these mice is randomized, indicating that Zic3 acts upstream of these genes in the determination of left-right asymmetry. The phenotype of these mice correctly models the defects found in human HTX1 and indicates an important role for Zic3 in both left-right and axial patterning.

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

Journal Article cDNA cloning of human hnRNP protein Al reveals the existence of multiple mRNA isoforms Get access M. Buvoli, M. Buvoli Search for other works by this author on: Oxford Academic PubMed Google Scholar G. Biamonti, G. Biamonti Search for other works by this author on: Oxford Academic PubMed Google Scholar P. Tsoulfas, P. Tsoulfas Search for other works by this author on: Oxford Academic PubMed Google Scholar M.T. Bassi, M.T. Bassi Search for other works by this author on: Oxford Academic PubMed Google Scholar A. Ghetti, A. Ghetti Search for other works by this author on: Oxford Academic PubMed Google Scholar S. Riva, S. Riva * * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar C. Morandi C. Morandi 1Istituto di Scienze Biologiche, Università degli Studi di VeronaItaly Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 16, Issue 9, 11 May 1988, Pages 3751–3770, https://doi.org/10.1093/nar/16.9.3751 Published: 11 May 1988 Article history Received: 02 February 1988 Revision received: 24 March 1988 Accepted: 24 March 1988 Published: 11 May 1988

The "ears of the lynx" MR imaging sign has been described in case reports of hereditary spastic paraplegia with a thin corpus callosum, mostly associated with mutations in the spatacsin vesicle trafficking associated gene, causing Spastic Paraplegia type 11 (SPG11). This sign corresponds to long T1 and T2 values in the forceps minor of the corpus callosum, which appears hyperintense on FLAIR and hypointense on T1-weighted images. Our purpose was to determine the sensitivity and specificity of the ears of the lynx MR imaging sign for genetic cases compared with common potential mimics.Four independent raters, blinded to the diagnosis, determined whether the ears of the lynx sign was present in each of a set of 204 single anonymized FLAIR and T1-weighted MR images from 34 patients with causal mutations associated with SPG11 or Spastic Paraplegia type 15 (SPG15). 34 healthy controls, and 34 patients with multiple sclerosis.The interrater reliability for FLAIR images was substantial (Cohen κ, 0.66-0.77). For these images, the sensitivity of the ears of the lynx sign across raters ranged from 78.8 to 97.0 and the specificity ranged from 90.9 to 100. The accuracy of the sign, measured by area under the receiver operating characteristic curve, ranged from very good (87.1) to excellent (93.9).The ears of the lynx sign on FLAIR MR imaging is highly specific for the most common genetic subtypes of hereditary spastic paraplegia with a thin corpus callosum. When this sign is present, there is a high likelihood of a genetic mutation, particularly associated with SPG11 or SPG15, even in the absence of a family history.

We took advantage of a large multinational recruitment to delineate genotype-phenotype correlations in a large, trans-European multicenter cohort of patients with spastic paraplegia gene 7 (SPG7).We analyzed clinical and genetic data from 241 patients with SPG7, integrating neurologic follow-up data. One case was examined neuropathologically.Patients with SPG7 had a mean age of 35.5 ± 14.3 years (n = 233) at onset and presented with spasticity (n = 89), ataxia (n = 74), or both (n = 45). At the first visit, patients with a longer disease duration (>20 years, n = 62) showed more cerebellar dysarthria (p < 0.05), deep sensory loss (p < 0.01), muscle wasting (p < 0.01), ophthalmoplegia (p < 0.05), and sphincter dysfunction (p < 0.05) than those with a shorter duration (<10 years, n = 93). Progression, measured by Scale for the Assessment and Rating of Ataxia evaluations, showed a mean annual increase of 1.0 ± 1.4 points in a subgroup of 30 patients. Patients homozygous for loss of function (LOF) variants (n = 65) presented significantly more often with pyramidal signs (p < 0.05), diminished visual acuity due to optic atrophy (p < 0.0001), and deep sensory loss (p < 0.0001) than those with at least 1 missense variant (n = 176). Patients with at least 1 Ala510Val variant (58%) were older (age 37.6 ± 13.7 vs 32.8 ± 14.6 years, p < 0.05) and showed ataxia at onset (p < 0.05). Neuropathologic examination revealed reduction of the pyramidal tract in the medulla oblongata and moderate loss of Purkinje cells and substantia nigra neurons.This is the largest SPG7 cohort study to date and shows a spasticity-predominant phenotype of LOF variants and more frequent cerebellar ataxia and later onset in patients carrying at least 1 Ala510Val variant.

<h2>Summary</h2> Although the best-known spinocerebellar ataxias (SCAs) are triplet repeat diseases, many SCAs are not caused by repeat expansions. The rarity of individual non-expansion SCAs, however, has made it difficult to discern genotype-phenotype correlations. We therefore screened individuals who had been found to bear variants in a non-expansion SCA-associated gene through genetic testing, and after we eliminated genetic groups that had fewer than 30 subjects, there were 756 subjects bearing single-nucleotide variants or deletions in one of seven genes: <i>CACNA1A</i> (239 subjects), <i>PRKCG</i> (175), <i>AFG3L2</i> (101), <i>ITPR1</i> (91), <i>STUB1</i> (77), <i>SPTBN2</i> (39), or <i>KCNC3</i> (34). We compared age at onset, disease features, and progression by gene and variant. There were no features that reliably distinguished one of these SCAs from another, and several genes—<i>CACNA1A</i>, <i>ITPR1</i>, <i>SPTBN2</i>, and <i>KCNC3—</i>were associated with both adult-onset and infantile-onset forms of disease, which also differed in presentation. Nevertheless, progression was overall very slow, and <i>STUB1</i>-associated disease was the fastest. Several variants in <i>CACNA1A</i> showed particularly wide ranges in age at onset: one variant produced anything from infantile developmental delay to ataxia onset at 64 years of age within the same family. For <i>CACNA1A</i>, <i>ITPR1</i>, and <i>SPTBN2</i>, the type of variant and charge change on the protein greatly affected the phenotype, defying pathogenicity prediction algorithms. Even with next-generation sequencing, accurate diagnosis requires dialogue between the clinician and the geneticist.

<h2>Abstract</h2><h3>Background</h3> Membrane Protein Palmitoylated 5 (<i>MPP5</i>) is a highly conserved apical complex protein, essential for cell polarity. Defects in neuronal cell polarity are associated with neurologic disorders. Only three patients with heterozygous <i>MPP5</i> de novo variants have been reported so far, with global developmental delay, behavioral changes and in only one case epileptic seizures. <h3>Objective</h3> To describe a new patient with a novel truncating de novo mutation in <i>MPP5</i> and to characterize in detail the epileptic phenotype and electroencephalographic features of the encephalopathy. <h3>Methods</h3> We identified a novel truncating de novo mutation in <i>MPP5</i> in a 44 year old patient by exome sequencing (p.Ser498Phefs*15). We retrospectively analyzed his clinical and instrumental data along a thirty-year follow up. <h3>Result</h3> Our patient presents with generalized tonic-clonic seizures, myoclonic and clonic seizures, non-epileptic myoclonus, tremor, severe intellectual disability, mild face dysmorphic traits, and psychosis. <h3>Discussion and conclusion</h3> We present a case of a childhood onset developmental encephalopathy with a likely-pathogenic variant in the <i>MPP5</i> gene.. This represents the first complete description of the epileptic syndrome associated with the MPP5 gene.

Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the protein's localization in the plasma membrane.

Research Article1 April 1990free access Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein. M. Buvoli M. Buvoli Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author F. Cobianchi F. Cobianchi Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author M.G. Bestagno M.G. Bestagno Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author A. Mangiarotti A. Mangiarotti Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author M.T. Bassi M.T. Bassi Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author G. Biamonti G. Biamonti Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author S. Riva S. Riva Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author M. Buvoli M. Buvoli Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author F. Cobianchi F. Cobianchi Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author M.G. Bestagno M.G. Bestagno Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author A. Mangiarotti A. Mangiarotti Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author M.T. Bassi M.T. Bassi Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author G. Biamonti G. Biamonti Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author S. Riva S. Riva Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. Search for more papers by this author Author Information M. Buvoli1, F. Cobianchi1, M.G. Bestagno1, A. Mangiarotti1, M.T. Bassi1, G. Biamonti1 and S. Riva1 1Istituto di Genetica Biochimica ed Evoluzionistica CNR, Pavia, Italy. The EMBO Journal (1990)9:1229-1235https://doi.org/10.1002/j.1460-2075.1990.tb08230.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human hnRNP core protein A1 (34 kd) is encoded by a 4.6 kb gene split into 10 exons. Here we show that the A1 gene can be differentially spliced by the addition of an extra exon. The new transcript encodes a minor protein of the hnRNP complex, here defined A1B protein, with a calculated mol. wt of 38 kd, that coincides with a protein previously designated as B2 by some authors. In vitro translation of the mRNAs selected by hybridization with A1 cDNA produced two proteins of 34 and 38 kd; Northern blot analysis of poly(A)+ RNA from HeLa cells revealed that the abundance of the A1B mRNA was approximately 5% that of A1. The A1B protein was detected by Western blotting with an anti-A1 monoclonal antibody both in enriched preparations of basic hnRNP proteins and in 40S hnRNP particles. The A1B protein exhibits a significantly higher affinity than A1 for ssDNA. The recombinant A1B protein, expressed in Escherichia coli, shows the same electrophoretic mobility and charge as the cellular one. Previous ArticleNext Article Volume 9Issue 41 April 1990In this issue RelatedDetailsLoading ...

Heterogeneous nuclear ribonucleoprotein (hnRNP) core protein A1 is a major component of mammalian hnRNP 40 S particles. We describe the structure of an active A1 gene and report on the partial characterization of the A1 gene family. About 30 A1-specific sequences are present per haploid human genome: 15 such sequences were isolated from a human genomic DNA library. Many corresponded to pseudogenes of the processed type but by applying a selection for actively transcribed regions we isolated an active A1 gene. The gene spans a region of 4.6 x 10(3) base-pairs and it is split into ten exons that encode the 320 amino acid residues of the protein. The amino acid sequence derived from the exon sequences is identical with that deduced from cDNA and reported for the protein. One intron exactly separates the two structural domains that constitute the protein. Each of the two RNA-binding domains in protein A1 is encoded by one exon. Experimental evidence indicates that the A1 gene can encode for more than one protein by alternative splicing. The gene is preceded by a strong promoter that contains at least two CCAAT boxes and two possible Sp1 binding sites, but it lacks a TATA box.

ZFYVE26/Spastizin and SPG11/Spatacsin encode 2 large proteins that are mutated in hereditary autosomal-recessive spastic paraplegia/paraparesis (HSP) type 15 (AR-SPG15) and type 11 (AR-SPG11), respectively. We previously have reported that AR-SPG15-related ZFYVE26 mutations lead to autophagy defects with accumulation of immature autophagosomes. ZFYVE26 and SPG11 were found to be part of a complex including the AP5 (adaptor related protein complex 5) and to have a critical role in autophagic lysosomal reformation with identification of autophagic and lysosomal defects in cells with both AR-SPG15- and AR-SPG11-related mutations. In spite of these similarities between the 2 proteins, here we report that ZFYVE26 and SPG11 are differently involved in autophagy and endocytosis. We found that both ZFYVE26 and SPG11 interact with RAB5A and RAB11, 2 proteins regulating endosome trafficking and maturation, but only ZFYVE26 mutations affected RAB protein interactions and activation. ZFYVE26 mutations lead to defects in the fusion between autophagosomes and endosomes, while SPG11 mutations do not affect this step and lead to a milder autophagy defect. We thus demonstrate that ZFYVE26 and SPG11 affect the same cellular physiological processes, albeit at different levels: both proteins have a role in autophagic lysosome reformation, but only ZFYVE26 acts at the intersection between endocytosis and autophagy, thus representing a key player in these 2 processes. Indeed expression of the constitutively active form of RAB5A in cells with AR-SPG15-related mutations partially rescues the autophagy defect. Finally the model we propose demonstrates that autophagy and the endolysosomal pathway are central processes in the pathogenesis of these complicated forms of hereditary spastic paraparesis.Abbreviations: ALR, autophagic lysosome reformation; AP5, adaptor related protein complex 5; AR, autosomal-recessive; HSP, hereditary spastic paraplegia/paraparesis; ATG14, autophagy related 14; BafA, bafilomycin A1; BECN1, beclin 1; EBSS, Earle balanced salt solution; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; HSP, hereditary spastic paraplegias; LBPA, lysobisphosphatidic acid; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MVBs, multivesicular bodies; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4, phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P, phosphatidylinositol-3-phosphate; RFP, red fluorescent protein; RUBCN, RUN and cysteine rich domain containing beclin 1 interacting protein; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; TCC: thin corpus callosum; TF, transferrin; UVRAG, UV radiation resistance associated.

Macroautophagy/autophagy is emerging as an important process in adult muscle stem cells functions: it regulates metabolic reprogramming during activation from a quiescent state, maintains stemness and prevents senescence. We now show that autophagy is specifically required for neonatal myogenesis and muscle development. Specific deletion of Atg7 in PAX7+ (paired box 7) precursors led in mice to a dwarf phenotype, with an effect restricted to the neonatal phase of muscle development. Atg7 knockdown suppressed neonatal satellite cell (nSC) proliferation and differentiation, downregulating the GH-IGF1 functions. When we disrupted autophagy, NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) accumulated in muscle and nSCs and negatively modulated DDIT3/CHOP (DNA-damage inducible transcript 3) expression. Lower levels of DDIT3 were responsible for reduced GHR expression leading to impaired local production of IGF1. Our results conclusively identify a novel autophagy-dependent pathway that regulates nSC behavior and indicate that autophagy is required for skeletal muscle development in the neonatal phase. Abbreviations: AKT/protein kinase B: Thymoma viral proto-oncogene; ASCs: adult stem cells; ATF4: activating transcription factor 4; ATG7: autophagy related 7; BAT: brown adipose tissue; BMP: bone morphogenetic protein; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CSA: cross sectional area; CTNNB1: catenin (cadherin associated protein), beta 1; DDIT3: DNA-damage inducible transcript 3; DM: differentiation medium; E: embryonic stage; EIF2AK3/PERK; EIF4EBP1: eukaryotic translation initiation factor 2 alpha kinase 3; eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; FGF21: fibroblast growth factor 21; GH: growth hormone; GHR: growth hormone receptor; HSCs: hematopoietic stem cells; IGF1: insulin-like growth factor 1; ITGAM: integrin alpha M; KEAP1: kelch-like ECH-associated protein 1; LY6A/Sca-1; MAP1LC3: lymphocyte antigen 6 complex, locus A; microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; miRNAs: microRNAs; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; mtUPR: mitochondrial unfolded protein response; MYF5: myogenic factor 5; MYH: myosin, heavy polypeptide; MYOD1: myogenic differentiation 1; MYOG: myogenin; NFE2L2: nuclear factor, erythroid derived 2, like 2; nSC: neonatal satellite cells; NSCs: neuronal stem cells; P: postnatal day; PAX7: paired box 7; PECAM1: platelet/endothelial cell adhesion molecule 1; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SCs: adult satellite cells; SQSTM1: sequestosome 1; STAT5: signal transducer and activator of transcription 5; TGFB1: transforming growth factor beta 1; WAT: white adipose tissue; WT: wild type.

Mitochondria play a critical role in neuronal function and neurodegenerative disorders, including Alzheimer's, Parkinson's and Huntington diseases and amyotrophic lateral sclerosis, that show mitochondrial dysfunctions associated with excessive fission and increased levels of the fission protein dynamin-related protein 1 (Drp1). Our data demonstrate that Drp1 regulates the transcriptional program induced by retinoic acid (RA), leading to neuronal differentiation. When Drp1 was overexpressed, mitochondria underwent remodeling but failed to elongate and this enhanced autophagy and apoptosis. When Drp1 was blocked during differentiation by overexpressing the dominant negative form or was silenced, mitochondria maintained the same elongated shape, without remodeling and this increased cell death. The enhanced apoptosis, observed with both fragmented or elongated mitochondria, was associated with increased induction of unfolded protein response (UPR) and ER-associated degradation (ERAD) processes that finally affect neuronal differentiation. These findings suggest that physiological fission and mitochondrial remodeling, associated with early autophagy induction are essential for neuronal differentiation. We thus reveal the importance of mitochondrial changes to generate viable neurons and highlight that, rather than multiple parallel events, mitochondrial changes, autophagy and apoptosis proceed in a stepwise fashion during neuronal differentiation affecting the nuclear transcriptional program.

The late-infantile-onset forms of neuronal ceroid lipofuscinosis (LINCL) are the most genetically heterogeneous group among the autosomal recessive neuronal ceroid lipofuscinoses (NCLs), with causative mutations found in CLN1, CLN2, CLN5, CLN6, CLN7 (MFSD8), and CLN8 genes. Homozygous mutations in CLN8 are associated with two distinct phenotypes: progressive epilepsy and mental retardation (EPMR), first identified in Finland; and a variant of late-infantile NCL (v-LINCL) described in a subset of Turkish and Italian patients. The function of the protein encoded by CLN8 is currently unknown. Here we report the identification of an Italian v-LINCL patient with a complete isodisomy of chromosome 8, leading to homozygosity of a maternally-inherited 3-bp deletion in CLN8 gene (c.180_182delGAA, p.Lys61del). Notably, uniparental disomy (UPD) has never been described associated with the NCLs. In addition, we provide evidence of the biological role of CLN8 characterized by expressing in different neuronal cell models the native protein, the protein carrying the mutation identified here, or three additional missense mutations previously described. Our results, validated through a gene silencing approach, indicate that CLN8 plays a role in cell proliferation during neuronal differentiation and in protection against cell death.

To gain a better understanding of the clinical and genetic features associated with agenesis of corpus callosum, we enrolled and characterized 162 patients with complete or partial agenesis of corpus callosum. Clinical and genetic protocols allowed us to categorize patients as syndromic subjects, affected by complex extra-brain malformations, and nonsyndromic subjects without any additional anomalies. We observed slight differences in sex ratio (56% males) and agenesis type (52% complete). Syndromic agenesis of corpus callosum subjects were prevalent (69%). We detected associated cerebral malformations in 48% of patients. Neuromotor impairment, cognitive and language disorders, and epilepsy were frequently present, regardless of the agenesis of corpus callosum subtype. Long-term follow-up allowed us to define additional indicators: syndromic agenesis of corpus callosum plus patients showed the most severe clinical features while isolated complete agenesis of corpus callosum patients had the mildest symptoms, although we observed intellectual disability (64%) and epilepsy (15%) in both categories. We achieved a definitive (clinical and/or genetic) diagnosis in 42% of subjects.

Mutations in tubulin genes are responsible for a large spectrum of brain malformations secondary to abnormal neuronal migration, organization, differentiation and axon guidance and maintenance. Motor impairment, intellectual disability and epilepsy are the main clinical symptoms. In the present study 15 patients from a personal cohort and 75 from 21 published studies carrying mutations in TUBA1A, TUBB2B and TUBB3 tubulin genes were evaluated with the aim to define a clinical and electrophysiological associated pattern. Epilepsy shows a wide range of severity without a specific pattern. Mutations in TUBA1A (60%) and TUBB2B (74%) and TUBB3 (25%) genes are associated with epilepsy. The accurate analysis of the Electroencephalogram (EEG) pattern in wakefulness and sleep in our series allows us to detect significant abnormalities of the background activity in 100% of patients. The involvement of white matter and of the inter-hemispheric connection structures typically observed in tubulinopathies is evidenced by the high percentage of asynchronisms in the organization of sleep activity recorded. In addition to asymmetries of the background activity, excess of slowing, low amplitude and Magnetic Resonance (MR) imaging confirm the presence of extensive brain malformations involving subcortical and midline structures. In conclusion, epilepsy in tubulinopathies when present has a favorable evolution over time suggesting a not particularly aggressive therapeutic approach.

Charcot-Marie-Tooth disease type 2A (CMT2A) is a rare inherited axonal neuropathy caused by mutations in MFN2 gene, which encodes Mitofusin 2, a transmembrane protein of the outer mitochondrial membrane. We performed a cross-sectional analysis on thirteen patients carrying mutations in MFN2, from ten families, describing their clinical and genetic characteristics. Evaluated patients presented a variable age of onset and a wide phenotypic spectrum, with most patients presenting a severe phenotype. A novel heterozygous missense variant was detected, p.K357E. It is located at a highly conserved position and predicted as pathogenic by in silico tools. At a clinical level, the p.K357E carrier shows a severe sensorimotor axonal neuropathy. In conclusion, our work expands the genetic spectrum of CMT2A, disclosing a novel mutation and its related clinical effect, and provides a detailed description of the clinical features of a cohort of patients with MFN2 mutations. Obtaining a precise genetic diagnosis in affected families is crucial both for family planning and prenatal diagnosis, and in a therapeutic perspective, as we are entering the era of personalized therapy for genetic diseases.

We recently identified a gene responsible for an autosomal recessive form of hereditary spastic paraplegia (HSP). This gene encodes paraplegin, a mitochondrial protein highly homologous to the yeast mitochondrial ATPases Afg3p and Rcalp, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane. By screening the Expressed Sequence Tag database, we identified and characterized a novel human cDNA, ATPase family gene 3-like 2 (AFG3L2, Human Gene Nomenclature Committee-approved symbol), which is closely related to paraplegin. This cDNA encodes a 797-amino-acid predicted protein highly similar to paraplegin as well as to yeast Afg3p and Rca1p. Immunofluorescence studies revealed that AFG3L2 and paraplegin share a similar expression pattern and the same subcellular localization, the mitochondrial compartment. We subsequently mapped AFG3L2 to chromosome 18p11 by radiation hybrid analysis. AFG3L2 may represent a candidate gene for other forms of HSPs and possibly for other neurodegenerative disorders.

Lombardia GENS is a multicentre prospective study aimed at diagnosing 5 single-gene disorders associated with stroke (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, Fabry disease, MELAS [mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes], hereditary cerebral amyloid angiopathy, and Marfan syndrome) by applying diagnostic algorithms specific for each clinically suspected diseaseWe enrolled a consecutive series of patients with ischemic or hemorrhagic stroke or transient ischemic attack admitted in stroke units in the Lombardia region participating in the project. Patients were defined as probable when presenting with stroke or transient ischemic attack of unknown etiopathogenic causes, or in the presence of <3 conventional vascular risk factors or young age at onset, or positive familial history or of specific clinical features. Patients fulfilling diagnostic algorithms specific for each monogenic disease (suspected) were referred for genetic analysis.In 209 patients (57.4±14.7 years), the application of the disease-specific algorithm identified 227 patients with possible monogenic disease. Genetic testing identified pathogenic mutations in 7% of these cases. Familial history of stroke was the only significant specific feature that distinguished mutated patients from nonmutated ones. The presence of cerebrovascular risk factors did not exclude a genetic disease.In patients prescreened using a clinical algorithm for monogenic disorders, we identified monogenic causes of events in 7% of patients in comparison to the 1% to 5% prevalence reported in previous series.

Mitochondrial dysfunction occurs in many muscle degenerative disorders. Here, we demonstrate that mitochondrial biogenesis was impaired in limb-girdle muscular dystrophy (LGMD) 2D patients and mice and was associated with impaired OxPhos capacity. Two distinct approaches that modulated histones or peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) acetylation exerted equivalent functional effects by targeting different mitochondrial pathways (mitochondrial biogenesis or fatty acid oxidation[FAO]). The histone deacetylase inhibitor Trichostatin A (TSA) changed chromatin assembly at the PGC-1α promoter, restored mitochondrial biogenesis, and enhanced muscle oxidative capacity. Conversely, nitric oxide (NO) triggered post translation modifications of PGC-1α and induced FAO, recovering the bioenergetics impairment of muscles but shunting the defective mitochondrial biogenesis. In conclusion, a transcriptional blockade of mitochondrial biogenesis occurred in LGMD-2D and could be recovered by TSA changing chromatin conformation, or it could be overcome by NO activating a mitochondrial salvage pathway.

The B-box family is an expanding new family of genes encoding proteins involved in diverse cellular functions such as developmental patterning and oncogenesis. A member of this protein family, MID1, is the gene responsible for the X-linked form of Opitz G/BBB syndrome, a developmental disorder characterized by defects of the midline structures. We now report the identification of MID2, a new transcript closely related to MID1. MID2 maps to Xq22 in human and to the syntenic region on the mouse X chromosome. The two X-linked genes share the same domains, the same exon-intron organization, a high degree of similarity at the protein level and the same subcellular localization, both being confined to the cytoplasm in association to micro-tubular structures. The expression pattern studied by RNA in situhybridization in mouse revealed that Mid2 is expressed early in development and the highest level of expression is detected in the heart, unlike Midi for which no expression was detected in the developing heart. Together, these data suggest that midin and MID2 have a similar biochemical function but a different physiological role during development.

The absorption coefficient of CuGe${\mathrm{O}}_{3}$ was measured in the energy range of 1-4 eV, in a temperature interval between 300 and 20 K, for light polarized along the $b$ and $c$ crystal axes. A weak absorption band at 1.7 eV is attributed to copper $d\ensuremath{-}d$ transitions partially allowed by electron-phonon interactions; this is followed by a region of transparency leading to a strong exponential edge above 3 eV, which is identified with the onset of charge-transfer transitions.

Opitz G/BBB Syndrome (OS) is a multiple congenital anomaly disorder characterized by defects along the body midline. The disease is characterized by variable expressivity of signs that include hypertelorism, cleft lip and/or palate, laryngo-tracheo-esophageal abnormalities, cardiac defects, and hypospadias. OS patients also present with mental retardation and brain anatomical abnormalities. An autosomal dominant form mapping to chromosome 22 and an X-linked form of OS are known. The gene responsible for the X-linked form of OS, MID1, codes for a member of the Tripartite Motif family of E3 ubiquitin ligases. Here we report 29 novel mutations in 29 unrelated patients of a cohort of 140 male OS cases. These mutations are found in both familial and sporadic cases. They are scattered along the entire length of the gene and are represented by missense and nonsense mutations, insertions and deletions causing frame shift mutations, and deletion of either single exons or the entire gene. The variety of the mutations found confirms that loss-of-function is the mechanism underlying the OS phenotype. Moreover, the low percentage of MID1-mutated OS patients, 47% of the familial and 13% of the sporadic cases, suggests a wider genetic heterogeneity underlying the OS phenotype. © 2007 Wiley-Liss, Inc.

Pontocerebellar hypoplasias (PCH) comprise a group of genetically heterogeneous disorders characterised by concurrent hypoplasia of the pons and the cerebellum and variable clinical and imaging features. The current classification includes 13 subtypes, with ~20 known causative genes. Attempts have been made to delineate the phenotypic spectrum associated to specific PCH genes, yet clinical and neuroradiological features are not consistent across studies, making it difficult to define gene-specific outcomes.We performed deep clinical and imaging phenotyping in 56 probands with a neuroradiological diagnosis of PCH, who underwent NGS-based panel sequencing of PCH genes and MLPA for CASK rearrangements. Next, we conducted a phenotype-based unsupervised hierarchical cluster analysis to investigate associations between genes and specific phenotypic clusters.A genetic diagnosis was obtained in 43 probands (77%). The most common causative gene was CASK, which accounted for nearly half cases (45%) and was mutated in females and occasionally in males. The European founder mutation p.Ala307Ser in TSEN54 and pathogenic variants in EXOSC3 accounted for 18% and 9% of cases, respectively. VLDLR, TOE1 and RARS2 were mutated in single patients. We were able to confirm only few previously reported associations, including jitteriness and clonus with TSEN54 and lower motor neuron signs with EXOSC3. When considering multiple features simultaneously, a clear association with a phenotypic cluster only emerged for EXOSC3.CASK represents the major PCH causative gene in Italy. Phenotypic variability associated with the most common genetic causes of PCH is wider than previously thought, with marked overlap between CASK and TSEN54-associated disorders.

Hereditary spastic paraplegias (HSPs) are a group of inherited rare neurologic disorders characterized by length-dependent degeneration of the corticospinal tracts and dorsal columns, whose prominent clinical feature is represented by spastic gait. Spastic paraplegia type 4 (SPG4, SPAST-HSP) is the most common form. We present both clinical and molecular findings of a large cohort of patients, with the aim of (1) defining the clinical spectrum of SPAST-HSP in Italy; (2) describing their molecular features; and (3) assessing genotype-phenotype correlations to identify features associated with worse disability.A cross-sectional retrospective study with molecular and clinical data collected in an anonymized database was performed.A total of 723 Italian patients with SPAST-HSP (58% men) from 316 families, with a median age at onset of 35 years, were included. Penetrance was 97.8%, with men showing higher Spastic Paraplegia Rating Scale (SPRS) scores (19.67 ± 12.58 vs 16.15 ± 12.61, p = 0.009). In 26.6% of patients with SPAST-HSP, we observed a complicated phenotype, mainly including intellectual disability (8%), polyneuropathy (6.7%), and cognitive decline (6.5%). Late-onset cases seemed to progress more rapidly, and patients with a longer disease course displayed a more severe neurologic disability, with higher SPATAX (3.61 ± 1.46 vs 2.71 ± 1.20, p < 0.001) and SPRS scores (22.63 ± 11.81 vs 12.40 ± 8.83, p < 0.001). Overall, 186 different variants in the SPAST gene were recorded, of which 48 were novel. Patients with SPAST-HSP harboring missense variants displayed intellectual disability (14.5% vs 4.4%, p < 0.001) more frequently, whereas patients with truncating variants presented more commonly cognitive decline (9.7% vs 2.6%, p = 0.001), cerebral atrophy (11.2% vs 3.4%, p = 0.003), lower limb spasticity (61.5% vs 44.5%), urinary symptoms (50.0% vs 31.3%, p < 0.001), and sensorimotor polyneuropathy (11.1% vs 1.1%, p < 0.001). Increasing disease duration (DD) and abnormal motor evoked potentials (MEPs) were also associated with increased likelihood of worse disability (SPATAX score>3).The SPAST-HSP phenotypic spectrum in Italian patients confirms a predominantly pure form of HSP with mild-to-moderate disability in 75% of cases, and slight prevalence of men, who appeared more severely affected. Early-onset cases with intellectual disability were more frequent among patients carrying missense SPAST variants, whereas patients with truncating variants showed a more complicated disease. Both longer DD and altered MEPs are associated with worse disability.

Hereditary spastic paraplegia (HSP) is a group of genetically heterogeneous disorders characterized by progressive spasticity of the lower limbs. Mutations in the SPG4 gene, which encodes spastin protein, are responsible for up to 45% of autosomal dominant cases.To search for disease-causing mutations in a large series of Italian patients with HSP.Samples of DNA were analyzed by direct sequencing of all exons in SPG4. Samples from a subset of patients were also analyzed by direct sequencing of all exons in SPG3A, SPG6, SPG10, and SPG13.Molecular testing facility in Italy.Sixty unrelated Italian patients with pure (n = 50) and complicated (n = 10) HSP.Mutations in SPG4, SPG3A, SPG6, SPG10, and SPG13.We identified 12 different mutations, 8 of which were novel, in 13 patients. No mutations of any of the other HSP genes tested were found in 15 patients with sporadic pure HSP who did not have mutations in the SPG4 gene.The overall rate of mutation in the SPG4 gene within our sample was 22%, rising to 26% when only patients with pure HSP were considered. The negative result obtained in 15 patients without mutations in SPG4 in whom 4 other genes were analyzed (SPG3A, SPG6, SPG10, and SPG13) indicate that these genes are not frequently mutated in sporadic pure HSP.

Abstract Autosomal recessive spastic paraplegia 52 is caused by biallelic mutations in AP4S1 which encodes a subunit of the adaptor protein complex 4 (AP‐4). Using next‐generation sequencing, we identified three novel unrelated SPG52 patients from a cohort of patients with cerebral palsy. The discovered variants in AP4S1 lead to reduced AP‐4 complex formation in patient‐derived fibroblasts. To further understand the role of AP4S1 in neuronal development and homeostasis, we engineered the first zebrafish model of AP‐4 deficiency using morpholino‐mediated knockdown of ap4s1 . In this model, we discovered several phenotypes mimicking SPG52, including altered CNS development, locomotor deficits, and abnormal neuronal excitability.

In the present study, we describe two novel cases of SCA5 with early onset. The first one, carrying a novel heterozygous de novo missense mutation in SPTBN2 gene, showed a striking very severe cerebellar atrophy and reduction of volume of the pons at a very young age (16 months). The latter, carrying the first de novo intragenic deletion so far reported in SPTBN2 gene, showed a mild cerebellar atrophy involving the hemispheres and a later onset. In both cases, for the first time, a hyperintense signal of the dentate nuclei was observed.

CAPN1 encodes calpain-1, a large subunit of μ-calpain, a calcium-activated cysteine protease widely present in the central nervous system. Mutations in CAPN1 have recently been identified in a complicated form of Hereditary Spastic Paraplegia (HSP) with a combination of cerebellar ataxia and corticomotor tract disorder (SPG76). Therefore, CAPN1 is now considered one of those genes that clinically manifest with a spectrum of disorders ranging from spasticity to cerebellar ataxia and represent a link between Spinocerebellar Ataxia and HSP, two groups of diseases previously considered separate but sharing pathophysiological pathways. We here describe clinical and molecular findings of two Italian adult siblings affected with a pure form of HSP and harboring the novel homozygote c.959delA variant (p.Tyr320Leufs*73) in the CAPN1 gene. Although the reason why mutations in CAPN1 may cause heterogeneous clinical pictures remains speculative, our findings confirm that the spectrum of the CAPN1-linked phenotypes includes pure HSP with onset during the third decade of life. Further studies are warrantied in order to clarify the mechanism underlying the differences in CAPN1 mutation clinical expression.

Defects in FARS2 are associated with either epileptic phenotypes or a spastic paraplegia subtype known as SPG77. Here, we describe an 8-year-old patient with severe and complicated spastic paraplegia, carrying a missense variant (p.Pro361Leu) and a novel intragenic deletion in FARS2. Of note, the disease is unexpectedly progressing rapidly and in a biphasic way differently from the previously reported cases. Our study provides the first detailed molecular characterization of a FARS2 deletion and its underlying molecular mechanism, and demonstrates the need for combining different tools to improve the diagnostic rate.

Some missense mutations and small deletions in the NOTCH3 gene, not involving cysteine residues, have been described in patients considered to be affected by paucisymptomatic CADASIL. However, the significance of such molecular variants is still unclear. We describe a 49-year-old woman with a CADASIL-like phenotype, carrying a novel cysteine-sparing mutation in exon 29 of the NOTCH3 gene, and discuss the possible pathogenetic role of this molecular variant. Even though atypical clinical and MRI findings make a diagnosis of CADASIL unlikely in this patient, our report nevertheless underlines the intriguing genotype-phenotype relationship in NOTCH3 mutations and the importance of functional investigation to ascertain the role of new NOTCH3 mutations in CADASIL pathogenesis.

Tensor-based morphometry (TBM) performed using T1-weighted images (T1WIs) is a well-established method for analyzing local morphological changes occurring in the brain due to normal aging and disease. However, in white matter regions that appear homogeneous on T1WIs, T1W-TBM may be inadequate for detecting changes that affect specific pathways. In these regions, diffusion tensor MRI (DTI) can identify white matter pathways on the basis of their different anisotropy and orientation. In this study, we propose performing TBM using deformation fields constructed using all scalar and directional information provided by the diffusion tensor (DTBM) with the goal of increasing sensitivity in detecting morphological abnormalities of specific white matter pathways. Previously, mostly fractional anisotropy (FA) has been used to drive registration in diffusion MRI-based TBM (FA-TBM). However, FA does not have the directional information that the tensors contain, therefore, the registration based on tensors provides better alignment of brain structures and better localization of volume change. We compare our DTBM method to both T1W-TBM and FA-TBM in investigating differences in brain morphology between patients with complicated hereditary spastic paraplegia of type 11 (SPG11) and a group of healthy controls. Effect size maps of T1W-TBM of SPG11 patients showed diffuse atrophy of white matter. However, DTBM indicated that atrophy was more localized, predominantly affecting several long-range pathways. The results of our study suggest that DTBM could be a powerful tool for detecting morphological changes of specific white matter pathways in normal brain development and aging, as well as in degenerative disorders.

Missense mutations within the kinesin family member 5A gene (KIF5A) are a known cause of a dominant form of hereditary spastic paraparesis (spastic paraplegia type 10- SPG10, OMIM: 604187) and of Charcot-Marie-Tooth disease type 2 (CMT2). Mutations in ALS2, the gene coding for alsin protein, have been demonstrated to be associated with a spectrum of rare autosomal recessive disorders including infantile ascending hereditary spastic paralysis (IAHSP), juvenile primary sclerosis (JPLS) with retrograde degeneration of the upper motor neurons, and juvenile ALS with involvement of both upper and lower motor neuron involvement. In this paper we describe a family in which the proband, a 14-year-old boy started manifesting an early onset (age 14 months) pure form of HSP rapidly progressing to a juvenile form of ALS. This boy carries a heterozygous missense variant in KIF5A, inherited from the father, and a homozygous missense variant in ALS2. The father, with family history of ALS, in the last few years has been developing signs and symptoms of affection of both the upper and lower motor neuron systems, with mild bulbar motor involvement and emotional lability. The patients described in this family, confirm the continuum and partial overlap of the two clinical entities, HSP and ALS, historically viewed as distinct entities. The genetic findings in this family further substantiate the genetic bases underlying the overlap, broadening the clinical spectrum associated with KIF5A mutations.

Translational activator of cytochrome c oxidase I (TACO1) is a mitochondrial translation factor involved in mitochondria-encoded cytochrome c oxidase subunit I (MT-CO1) synthesis.1,2 Loss-of-function mutations in the TACO1 gene cause respiratory chain complex IV deficiency. Clinically heterogeneous human diseases are due to cytochrome c oxidase (COX) deficiency, ranging from Leigh syndrome to myopathy, deafness, or ataxia. Recently, 2 different TACO1 mutations have been identified in 3 families with late-onset Leigh syndrome and a leukoencephalopathy involving predominantly basal ganglia and cystic changes.3,4 Here, we report a subject carrying a novel homozygous truncating mutation in the TACO1 gene and presenting an adult-onset slowly progressive spastic paraparesis with cognitive impairment and a subcortical U-fiber leukoencephalopathy. The authors thank the patient and her family for participating in the study and Valentina Baderna for the technical support in functional studies.

Alport's syndrome is characterized clinically by a nonimmune glomerulopathy, often accompanied by sensorineural hearing loss and lens abnormalities, frequently due to mutations in the COL4A5 gene. The association of AS with diffuse leiomyomatosis, a benign proliferation of smooth muscle that occurs most often in the esophagus, trachea, and female genitalia, has been reported. Recently, a deletion involving both the COL4A5 and COL4A6 genes has been reported in four unrelated families. We report an additional case with Alport's syndrome associated with leiomyomatosis carrying a deletion of both COL4A5 and COL4A6 genes. A detailed characterization of the genomic region involved in the deletion event has been performed. Our results demonstrate that the deletion removed exon 1 of COL4A5 and exons 1 and 2 of COL4A6.

Abstract Septo-optic dysplasia (SOD) syndrome is a rare congenital disorder characterized by a classic triad of optic nerve/chiasm hypoplasia, agenesis of septum pellucidum and corpus callosum, and hypoplasia of the hypothalamic-pituitary axis. Herein, we report the clinical case of 2-year-old boy presenting with psychomotor delay, nystagmus, congenital hypothyroidism, and a clinically relevant growth delay. The neuroradiological examination showed partial segmental agenesis of the corpus callosum, agenesis of the septum pellucidum, optic nerve hypoplasia, and a small pituitary gland with a small median pituitary stalk. A whole-exome sequencing analysis detected a novel heterozygous de novo variant c.1069_1070delAG in SON, predicted as likely pathogenic. To date, SON pathogenic variants have been described as responsible for Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome, a multisystemic neurodevelopmental disorder mainly characterized by intellectual disability, facial dysmorphisms, visual abnormalities, brain malformations, feeding difficulties, and growth delay. The herein described case is the first recognized clinic-radiological occurrence of SOD syndrome with hypothalamic-pituitary dysfunction in a patient carrying a SON gene variant, considered responsible of ZTTK syndrome, suggesting a possible relationship between SOD and SON gene alterations, never described so far, making the search for SON gene mutations advisable in patients with SOD.

X-linked adrenoleukodystrophy (x-ALD) is a rare genetic disorder caused by a mutation in the ABCD1 gene, which encodes for a peroxisomal very long chain fatty acid transporter. Clinically, x-ALD can present a wide spectrum of different phenotypes: asymptomatic carriers, Addison only, cerebral x-ALD, and myelopathy with/without evidence of peripheral axonopathy (Adrenomyeloneuropathy). We report on two cases of adult x-ALD, with atypical phenotypes: (Case 1) A 37-years-old male with a 2-years-long history of spastic paraparesis, urinary urgency, and subclinical adrenocortical insufficiency. As an atypical finding, the MRI showed multiple congenital brain development defects. (Case 2) A 63-years-old male with a previous diagnosis of Addison disease, with a 6-years-long history of spastic paraparesis. Two years later, he complained of severe and disabling burning pain in his feet. A nerve conduction study was normal, but a skin biopsy revealed autonomic and somatic small fiber neuropathy. In both cases, genetic testing disclosed hemizygous mutation in ABCD1 associated with x-ALD: c.1394-2A > G and p.(Thr254Met), respectively. While case 1 supports the key role of peroxisome functions in brain development, case 2 points to a possible selective and clinically relevant peripheral small fiber degeneration in x-ALD myelopathy.

This long-term retrospective follow-up study aimed to address the knowledge gap between prenatal diagnosis of complete isolated Agenesis of Corpus Callosum (cACC) at fetal MRI and postnatal neurodevelopmental outcome to improve prenatal counseling for parents.Data on fetuses with isolated cACC from a single-center MRI database built up in two decades were considered. Detailed postnatal clinical, neuropsychological evaluations were performed and descriptions of available neuroradiological and genetic data were provided.Following a detailed neuropsychological evaluation and a long-term follow-up, the subsequent results emerged: 38 school-aged children (older than 6 years) of 50 (aged 2.5-15 years) showed normal intellectual functions (50%), intellectual disability (21%), and borderline intelligence quotient (29%). Deficits in motor functions (58%), executive functions (37%), language (61%), memory abilities (58%), and academic performances (53%) were found. Twenty-one percent of participants showed behavioral difficulties. Almost half of the participants underwent rehabilitation. Additional findings (21%) were detected at postnatal brain MRI, and a significant association between additional findings at postnatal imaging and abnormal neurodevelopmental outcome was observed.This study supports the view that children with prenatal diagnosis of isolated cACC may present with several degrees of neurologic and neuropsychological impairment which become more evident only in their second decade of life. Postnatal MRI and detailed genetic analysis may add crucial information to prenatal data and substantially influence final judgment on the outcome and orient clinical management and counseling.

Dynamin-1-like (DNM1L) is a gene located on chromosome 12p11.21 that encodes for dynamin-related protein (DRP1), a GTPase involved in mitochondrial and peroxisomal fusion, which plays a pivotal role in brain development. The missense variant, p.Arg403Cys, is clinically associated with childhood-onset super-refractory status epilepticus, with either subsequent poor neurological outcome or death (described in 13 patients). We present a 20-year-old girl carrying this mutation with a history of two episodes of super-refractory focal myoclonic status epilepticus which manifested as epilepsia partialis continua (EPC) with a 13-year interval, during which she displayed moderate intellectual disability, social and school reintegration, without complete control of myoclonic manifestations. The first status, which occurred at the age of six, was associated with transient left side thalamic involvement and the second episode with right side transient basal ganglia hyperintensity on MRI. After the second status, a persistent vegetative state with both drug-resistant epilepsia partialis continua and reticular myoclonus endured; the MRI showed progressive brain atrophy. In contrast to previous published cases, this new case of childhood-onset DNM1L encephalopathy demonstrated biphasic clinical progression. The main features of our patient were EPC, super-refractory status epilepticus, and transient and migrating subcortical thalamic hyperintensity on MRI at onset. The unusual clinical course is also noticeable, indicating possible epigenetic and/or protective factors, without underestimating the progressive and genetic basis of this encephalopathy. Precise characterization of seizures and whole-exome sequencing are crucial in order to establish early diagnosis.

Abstract SPG15 is a hereditary spastic paraplegia subtype caused by mutations in Spastizin, a protein encoded by the ZFYVE26 gene. Spastizin is involved in autophagosome maturation and autophagic lysosome reformation and SPG15-related mutations lead to autophagic lysosome reformation defects with lysosome enlargement, free lysosome depletion and autophagosome accumulation. Symptomatic and rehabilitative treatments are the only therapy currently available for patients. Here, we targeted autophagy and lysosomes in SPG15 patient-derived cells by using a library of autophagy-modulating compounds. We identified a rose of compounds affecting intracellular calcium levels, the calcium-calpain pathway or lysosomal functions, which reduced autophagosome accumulation. The six most effective compounds were tested in vivo in a new SPG15 loss of function Drosophila model that mimicked the reported SPG15 phenotype, with autophagosome accumulation, enlarged lysosomes, reduced free lysosomes, autophagic lysosome reformation defects and locomotor deficit. These compounds, namely verapamil, Bay K8644, 2′,5′-dideoxyadenosine, trehalose, Small-Molecule Enhancer of Rapamycin 28 and trifluoperazine, improved lysosome biogenesis and function in vivo, demonstrating that lysosomes are a key pharmacological target to rescue SPG15 phenotype. Among the others, the Small-Molecule Enhancer of Rapamycin 28 was the most effective, rescuing both autophagic lysosome reformation defects and locomotor deficit, and could be considered as a potential therapeutic compound for this hereditary spastic paraplegia subtype.

Journal Article A submicroscopic deletion in a patient with isolated X-linked ocular albinism (OA1) Get access Maria T. Bassi, Maria T. Bassi Search for other works by this author on: Oxford Academic PubMed Google Scholar Arthur A.B. Bergen, Arthur A.B. Bergen 1The Netherlands Ophthalmic Research InstituteAmsterdam, The Netherlands Search for other works by this author on: Oxford Academic PubMed Google Scholar Martin C. Wapenaar, Martin C. Wapenaar Search for other works by this author on: Oxford Academic PubMed Google Scholar Maria V. Schlaffino, Maria V. Schlaffino Search for other works by this author on: Oxford Academic PubMed Google Scholar Mary van Schooneveld, Mary van Schooneveld 1The Netherlands Ophthalmic Research InstituteAmsterdam, The Netherlands Search for other works by this author on: Oxford Academic PubMed Google Scholar John R.W. Yates, John R.W. Yates 2Department of Clinical Genetics, Addenbrooke's HospitalCambridge Search for other works by this author on: Oxford Academic PubMed Google Scholar Stephen J. Charles, Stephen J. Charles 3The Royal Eye HospitalManchester, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Thomas Meltinger, Thomas Meltinger 4Abteilung fūr Padiatrische Genetik, Kinderpoliklinik der Ludwig-Maximilians-UniversrtātMūnchen, Germany Search for other works by this author on: Oxford Academic PubMed Google Scholar Andrea Ballabio Andrea Ballabio * *To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Human Molecular Genetics, Volume 3, Issue 4, April 1994, Pages 647–648, https://doi.org/10.1093/hmg/3.4.647 Published: 01 April 1994 Article history Received: 29 November 1993 Accepted: 08 February 1994 Published: 01 April 1994

Heterogeneous nuclear ribonucleoprotein (HNRP) core protein A1 is a major component of mammalian HNRP particles. The human HNRP A1 protein was shown to be encoded by a 4.6-kb gene, split into 10 exons, belonging to a multigene family of about 30 A1-specific sequences per haploid genome, many of which correspond to pseudogenes of the processed type. Here we report the mapping of the human HNRPA1 gene to band 12q13.1. Localization was performed by nonisotopic in situ hybridization using a phage genomic clone that contains the active HNRPA1 gene as well as 13.5-kb flanking sequences. To suppress hybridization to pseudogene sequences, unlabeled HNRPA1 cDNA was added in excess over the probe to the hybridization mixture.

Childhood-onset peripheral neuropathies are often of genetic origin. Charcot-Marie-Tooth (CMT), is considered the commonest neuromuscular disorder. Due to its high clinical heterogeneity, especially in the pediatric age, the co-existence of central and peripheral symptoms and signs does not necessarily rule out a diagnosis of hereditary peripheral neuropathy.We describe the clinical, neurophysiological and genetic findings in a teen-age patient evaluated for acquired toe-walking and progressive difficulties in walking since the age of 5. Genetic testing was carried out with a targeted NGS panel. Identified variants are analyzed using Variant Studio program (Illumina). Rare variants and variants considered as pathogenic were analyzed by Sanger direct sequencing.The coexistence of peripheral and pyramidal signs in the lower limbs, the absence of a significant pre/perinatal history, the unremarkable brain and spine MRI, together with the presence of a sensory-motor polyneuropathy in all four limbs, prompted the execution of genetic investigations with an NGS panel covering hereditary spastic paraplegias, motor neuron disease and Charcot-Marie-Tooth. We identified a previously undescribed variant (c.1142G>T, p.Arg381Leu) in the EGR2 gene.ERG2 gene has been described as a cause of various phenotypes, including a rare autosomal dominant form of CMT (CMT type 1D) representing approximately 1% of all CMT subgroups. We describe a novel pathogenic variant in EGR2 gene leading to the development of a complex association of peripheral and central neurological signs, underscoring the genetic and clinical heterogeneity of hereditary neuropathies of pediatric onset.

Mutations in spastic paraplegia gene 7 (SPG7) are associated with a variable phenotype, including spastic paraplegia, cerebellar ataxia, progressive external ophthalmoplegia, cognitive impairment, neuropathy, muscle wasting, optic atrophy, deafness, parkinsonism and epilepsy [ [1] Coarelli G. Schule R. van de Warrenburg B.P.C. De Jonghe P. Ewenczyk C. Martinuzzi A. et al. Loss of paraplegin drives spasticity rather than ataxia in a cohort of 241 patients with SPG7. Neurology. 2019; 92 (e2679–e2690) Crossref PubMed Scopus (24) Google Scholar , [2] De la Casa-Fages B. Fernández-Eulate G. Gamez J. Barahona-Hernando R. Morís G. García-Barcina M. et al. Parkinsonism and spastic paraplegia type 7: expanding the spectrum of mitochondrial Parkinsonism. Mov. Disord. 2019; 34: 1547-1561 Crossref PubMed Scopus (18) Google Scholar ]. SPG7 encodes paraplegin, located in mitochondria and in the endoplasmatic reticulum and is involved in mitochondrial functions and axonal development [ [3] Atorino L. Silvestri L. Koppen M. Cassina L. Ballabio A. Marconi R. et al. Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. J. Cell Biol. 2003; 163: 777-787 Crossref PubMed Scopus (201) Google Scholar , [4] Mancuso G. Barth E. Crivello P. Rugarli E.I. Alternative splicing of Spg7, a gene involved in hereditary spastic paraplegia, encodes a variant of paraplegin targeted to the endoplasmic reticulum. PloS One. 2012; 7e36337 Crossref PubMed Scopus (7) Google Scholar ]. It is an adenosine triphosphate (ATP)-dependent zinc metalloproteinase assembling with the homologous subunit AFG3L2 into a mitochondrial matrix quality control protease complex [ [3] Atorino L. Silvestri L. Koppen M. Cassina L. Ballabio A. Marconi R. et al. Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia. J. Cell Biol. 2003; 163: 777-787 Crossref PubMed Scopus (201) Google Scholar ]. Mutations in AFG3L2 gene cause spinocerebellar ataxia 28 or spastic ataxia type 5. Recently, four siblings with two novel compound heterozygous variants in the AFG3L2 gene has been reported to have an eye-of-the-tiger (EOT)-like sign on MRI [ [5] Calandra C.R. Buda G. Vishnopolska S.A. Oliveri J. Olivieri F.A. Pérez Millán M.I. et al. Spastic ataxia with eye-of-the-tiger-like sign in 4 siblings due to novel compound heterozygous AFG3L2 mutation. Park. Relat. Disord. 2020; 73: 52-54 Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar ]. They had a T2-weighted central hyperintense signal in the anteromedial portion of both globus pallidus, similarly to the sign reported in pantothenate kinase-associated neurodegeneration, but without the surrounding low signal intensity due to excessive iron deposition [ [5] Calandra C.R. Buda G. Vishnopolska S.A. Oliveri J. Olivieri F.A. Pérez Millán M.I. et al. Spastic ataxia with eye-of-the-tiger-like sign in 4 siblings due to novel compound heterozygous AFG3L2 mutation. Park. Relat. Disord. 2020; 73: 52-54 Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar ]. Given the close link between AFG3L2 and paraplegin, we search for a similar MRI sign in five unrelated patients with mutations in SPG7.

Abstract The continuous emergence of SARS-CoV-2 variants of concern with mutated spike receptor binding domains has rendered many therapeutic mAbs ineffective. To date, there are no clinically authorized therapeutic antibodies effective against the predominant circulating sub-lineages BQ and XBB. Here, we report the isolation of broad and potent neutralizing HuMabs from a Danish healthcare worker infected with SARS-CoV-2 early in the pandemic. These HuMabs include a novel and genetically unique non-RBD-specific HuMab (K501SP6) which can neutralize Omicron sub-lineages BQ and XBB, and an RBD-specific HuMab (K501SP3) with high potency towards earlier circulating variants but was escaped by Omicron sub-lineages BA.5, BQ and XBB through F486 and E484 substitutions. Characterizing SARS-CoV-2 spike-specific HuMabs, including broadly reactive non-RBD-specific HuMabs, can give insight into the immune mechanisms involved in neutralization and immune evasion, which can be a valuable addition to already existing SARS-CoV-2 therapies.

Objective: To determine the sensitivity and specificity for SPG11-15 mutations of an MRI abnormality in the forceps minor of the corpus callosum (“ears of the lynx” sign). Background: The “Ears of the Lynx” sign has been described in patients with hereditary spastic paraparesis associated with mutations of the SPG11 gene (Riverol 2009). It consists of an abnormality of the forceps minor of the corpus callosum, corresponding to the genu fibers, which appears bright on T2-FLAIR and dark on T1-weighted images. Design/Methods: We studied T1-weighted and T2-FLAIR MR images from 24 patients with SPG mutations (18 in SPG11, 2 in SPG15, 2 in SPG7 and 2 in SPG4), 24 disease controls with multiple sclerosis (MS), and 24 healthy controls matched by age and sex. The MS group was used as a comparison group as it is the disorder most likely to cause white matter changes in the age group under consideration. MRI images were read and classified by two independent observers, blind to the clinical picture. Results: The “Ears of the Lynx” sign was present only in SPG11 and SPG15 mutations. Both sensitivity and specificity were higher for the T2-FLAIR (94[percnt] and 97[percnt]) than for the T1-weighted images (81[percnt] and 94[percnt]). There was a high degree of concordance between the two observers for the T2-FLAIR images (kappa = 0.89) but not for the T1- weighted images (kappa = 0.60). Conclusions/Relevance: In this sample, the “Ear of the Lynx” sign on T2-FLAIR MRI separated well patients with SPG11 and SPG15 mutations from patients with other SPG mutations, multiple sclerosis and healthy controls. As it was present early in the course of the disease, it may be a helpful indication of neurological involvement in subjects at risk. Disclosure: Dr. Masdeu has received personal compensation in an editorial capacity for the Journal of Neuroimaging. Dr. Pascual has nothing to disclose. Dr. Franca, Jr. has nothing to disclose. Dr. Gregory has nothing to disclose. Dr. Daniels has nothing to disclose. Dr. Patronas has nothing to disclose. Dr. Toro has nothing to disclose. Dr. Bassi has nothing to disclose. Dr. Charles Ignatiew has nothing to disclose.

Filamin C is a large dimeric actin-binding protein, most prevalent in skeletal and cardiac muscle Z-discs, where it participates in sarcomere mechanical stabilization and intracellular signaling, interacting with numerous binding partners. Dominant heterozygous mutations of Filamin C gene cause several forms of myopathy and structural or arrhythmogenic cardiomyopathy. In this report we describe clinical and molecular findings of two Italian patients, in whom we identified two novel missense variants located within the Filamin C actin binding domain. Muscle imaging, histological and ultrastructural findings are also reported. Our results underline the extreme inter- and intrafamilial variability of clinical manifestations, hence the need to extend the investigation also to asymptomatic relatives, and the relevance of a broad diagnostic approach involving muscle electron microscopy, skeletal muscle magnetic resonance imaging and next generation sequencing techniques.

Raw data from the research article "Rescue of lysosomal function as therapeutic strategy for SPG15 hereditary spastic paraplegia".

Raw data from the research article "Rescue of lysosomal function as therapeutic strategy for SPG15 hereditary spastic paraplegia".

Macroautophagy/autophagy is emerging as an important process in adult muscle stem cells functions: it regulates metabolic reprogramming during activation from a quiescent state, maintains stemness and prevents senescence. We now show that autophagy is specifically required for neonatal myogenesis and muscle development. Specific deletion of <i>Atg7</i> in PAX7⁺ (paired box 7) precursors led in mice to a dwarf phenotype, with an effect restricted to the neonatal phase of muscle development. <i>Atg7</i> knockdown suppressed neonatal satellite cell (nSC) proliferation and differentiation, downregulating the GH-IGF1 functions. When we disrupted autophagy, NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) accumulated in muscle and nSCs and negatively modulated DDIT3/CHOP (DNA-damage inducible transcript 3) expression. Lower levels of DDIT3 were responsible for reduced GHR expression leading to impaired local production of IGF1. Our results conclusively identify a novel autophagy-dependent pathway that regulates nSC behavior and indicate that autophagy is required for skeletal muscle development in the neonatal phase. <b>Abbreviations:</b> AKT/protein kinase B: Thymoma viral proto-oncogene; ASCs: adult stem cells; ATF4: activating transcription factor 4; ATG7: autophagy related 7; BAT: brown adipose tissue; BMP: bone morphogenetic protein; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CSA: cross sectional area; CTNNB1: catenin (cadherin associated protein), beta 1; DDIT3: DNA-damage inducible transcript 3; DM: differentiation medium; E: embryonic stage; EIF2AK3/PERK; EIF4EBP1: eukaryotic translation initiation factor 2 alpha kinase 3; eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; FGF21: fibroblast growth factor 21; GH: growth hormone; GHR: growth hormone receptor; HSCs: hematopoietic stem cells; IGF1: insulin-like growth factor 1; ITGAM: integrin alpha M; KEAP1: kelch-like ECH-associated protein 1; LY6A/Sca-1; MAP1LC3: lymphocyte antigen 6 complex, locus A; microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; miRNAs: microRNAs; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; mtUPR: mitochondrial unfolded protein response; MYF5: myogenic factor 5; MYH: myosin, heavy polypeptide; MYOD1: myogenic differentiation 1; MYOG: myogenin; NFE2L2: nuclear factor, erythroid derived 2, like 2; nSC: neonatal satellite cells; NSCs: neuronal stem cells; P: postnatal day; PAX7: paired box 7; PECAM1: platelet/endothelial cell adhesion molecule 1; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SCs: adult satellite cells; SQSTM1: sequestosome 1; STAT5: signal transducer and activator of transcription 5; TGFB1: transforming growth factor beta 1; WAT: white adipose tissue; WT: wild type.

Macroautophagy/autophagy is emerging as an important process in adult muscle stem cells functions: it regulates metabolic reprogramming during activation from a quiescent state, maintains stemness and prevents senescence. We now show that autophagy is specifically required for neonatal myogenesis and muscle development. Specific deletion of <i>Atg7</i> in PAX7⁺ (paired box 7) precursors led in mice to a dwarf phenotype, with an effect restricted to the neonatal phase of muscle development. <i>Atg7</i> knockdown suppressed neonatal satellite cell (nSC) proliferation and differentiation, downregulating the GH-IGF1 functions. When we disrupted autophagy, NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) accumulated in muscle and nSCs and negatively modulated DDIT3/CHOP (DNA-damage inducible transcript 3) expression. Lower levels of DDIT3 were responsible for reduced GHR expression leading to impaired local production of IGF1. Our results conclusively identify a novel autophagy-dependent pathway that regulates nSC behavior and indicate that autophagy is required for skeletal muscle development in the neonatal phase. <b>Abbreviations:</b> AKT/protein kinase B: Thymoma viral proto-oncogene; ASCs: adult stem cells; ATF4: activating transcription factor 4; ATG7: autophagy related 7; BAT: brown adipose tissue; BMP: bone morphogenetic protein; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CSA: cross sectional area; CTNNB1: catenin (cadherin associated protein), beta 1; DDIT3: DNA-damage inducible transcript 3; DM: differentiation medium; E: embryonic stage; EIF2AK3/PERK; EIF4EBP1: eukaryotic translation initiation factor 2 alpha kinase 3; eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; FGF21: fibroblast growth factor 21; GH: growth hormone; GHR: growth hormone receptor; HSCs: hematopoietic stem cells; IGF1: insulin-like growth factor 1; ITGAM: integrin alpha M; KEAP1: kelch-like ECH-associated protein 1; LY6A/Sca-1; MAP1LC3: lymphocyte antigen 6 complex, locus A; microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; miRNAs: microRNAs; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; mtUPR: mitochondrial unfolded protein response; MYF5: myogenic factor 5; MYH: myosin, heavy polypeptide; MYOD1: myogenic differentiation 1; MYOG: myogenin; NFE2L2: nuclear factor, erythroid derived 2, like 2; nSC: neonatal satellite cells; NSCs: neuronal stem cells; P: postnatal day; PAX7: paired box 7; PECAM1: platelet/endothelial cell adhesion molecule 1; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SCs: adult satellite cells; SQSTM1: sequestosome 1; STAT5: signal transducer and activator of transcription 5; TGFB1: transforming growth factor beta 1; WAT: white adipose tissue; WT: wild type.

Hereditary spastic paraplegias (HSPs) are rare, inherited neurological disorders, characterised by degeneration of the corticospinal tracts and dorsal columns. SPG4, caused by heterozygous mutations of the SPAST gene, represents the most common form. Herein, we present findings from the largest described cohort of Italian SPG4 patients, aiming to define the clinical spectrum of SPG4 in Italy, and find any significant genotype-phenotype correlations.