Novel loss of function mutation in TUBA1A gene compromises tubulin stability and proteostasis causing spastic paraplegia and ataxia.

Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and ß-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific α-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for α-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in TUBA1A, and expand both phenotypic and mutational spectrum related to this gene.

Deciphering the Tubulin Language: Molecular Determinants and Readout Mechanisms of the Tubulin Code in Neurons.

Microtubules (MTs) are dynamic components of the cell cytoskeleton involved in several cellular functions, such as structural support, migration and intracellular trafficking. Despite their high similarity, MTs have functional heterogeneity that is generated by the incorporation into the MT lattice of different tubulin gene products and by their post-translational modifications (PTMs). Such regulations, besides modulating the tubulin composition of MTs, create on their surface a "biochemical code" that is translated, through the action of protein effectors, into specific MT-based functions. This code, known as "tubulin code", plays an important role in neuronal cells, whose highly specialized morphologies and activities depend on the correct functioning of the MT cytoskeleton and on its interplay with a myriad of MT-interacting proteins. In recent years, a growing number of mutations in genes encoding for tubulins, MT-interacting proteins and enzymes that post-translationally modify MTs, which are the main players of the tubulin code, have been linked to neurodegenerative processes or abnormalities in neural migration, differentiation and connectivity. Nevertheless, the exact molecular mechanisms through which the cell writes and, downstream, MT-interacting proteins decipher the tubulin code are still largely uncharted. The purpose of this review is to describe the molecular determinants and the readout mechanisms of the tubulin code, and briefly elucidate how they coordinate MT behavior during critical neuronal events, such as neuron migration, maturation and axonal transport.

Neurological and Neuroimaging Features of CYB5R3-Related Recessive Hereditary Methemoglobinemia Type II.

Recessive hereditary methemoglobinemia (RHM) due to NADH-cytochrome b5 reductase deficiency is a rare disease caused by pathogenic variants in CYB5R3. Unlike type I, in RHM type II (RHM2), the enzymatic defect affects erythrocytes and all body tissues, thus resulting in cyanosis and neurological impairment. Although the first description of RHM2 dates back to the mid-1950s, detailed clinical and neuroimaging information are available for only a few patients. Here, we describe a new patient with RHM2 that harbors an unreported homozygous 31 Kb deletion involving part of CYB5R3, and showing a peculiar neuroimaging pattern resembling a ponto-cerebellar hypoplasia-like condition. A careful review of the available literature was performed with the aim of better delineating neurological and neuroimaging as well as the genotypic spectra of this extremely rare disease.

Broadening the spectrum phenotype of TBCE-related neuron neurodegeneration.

BACKGROUND: Severe loss of TBCE function has been related to two well-known dysmorphic syndromes, while TBCE hypomorphic variants have been linked to neurodegenerative conditions due to perturbed microtubule dynamics and homeostasis, with signs of central and peripheral nervous system involvement. METHOD: We report on an Italian female originating from Southern Italy who presented early-onset regression and neurodegeneration, with neurological features of tetraparesis and signs of peripheral nervous system involvement. Her brain MRI revealed white matter involvement. RESULTS: Analyzing all known hypomyelination leukodystrophies related genes, two mutations in TBCE (NM_001079515) were detected: the missense variant c.464 T > A; p. (Ile155Asn) and the frameshift variant c.924del; p. (Leu309Ter), in compound heterozygosity, already reported in the literature in patients coming from the same geographical area. The clinical phenotype of the proposita was more severe and with an earlier onset than the majority of the patients reported so far. CONCLUSIONS: Next Generation Sequencing is becoming increasingly necessary to assess unusual phenotypes, with the opportunity to establish prognosis and disease mechanisms, and facilitating differential diagnosis.

Biallelic mutations in RNF220 cause laminopathies featuring leukodystrophy, ataxia and deafness.

Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report these two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the underlying cause of this novel form of leukodystrophy with ataxia and sensorineural deafness that includes fibrotic cardiomyopathy and hepatopathy as associated features in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as the RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology; mutations in primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.

Heterozygous KIF1A variants underlie a wide spectrum of neurodevelopmental and neurodegenerative disorders.

BACKGROUND: Dominant and recessive variants in the KIF1A gene on chromosome 2q37.3 are associated with several phenotypes, although only three syndromes are currently listed in the OMIM classification: hereditary sensory and autonomic neuropathy type 2 and spastic paraplegia type 30, both recessively inherited, and mental retardation type 9 with dominant inheritance. METHODS: In this retrospective multicentre study, we describe the clinical, neuroradiological and genetic features of 19 Caucasian patients (aged 3-65 years) harbouring heterozygous KIF1A variants, and extensively review the available literature to improve current classification of KIF1A-related disorders. RESULTS: Patients were divided into two groups. Group 1 comprised patients with a complex phenotype with prominent pyramidal signs, variably associated in all but one case with additional features (ie, epilepsy, ataxia, peripheral neuropathy, optic nerve atrophy); conversely, patients in group 2 presented an early onset or congenital ataxic phenotype. Fourteen different heterozygous missense variants were detected by next-generation sequencing screening, including three novel variants, most falling within the kinesin motor domain. CONCLUSION: The present study further enlarges the clinical and mutational spectrum of KIF1A-related disorders by describing a large series of patients with dominantly inherited KIF1A pathogenic variants ranging from pure to complex forms of hereditary spastic paraparesis/paraplegias (HSP) and ataxic phenotypes in a lower proportion of cases. A comprehensive review of the literature indicates that KIF1A screening should be implemented in HSP regardless of its mode of inheritance or presentations as well as in other complex neurodegenerative or neurodevelopmental disorders showing congenital or early onset ataxia.

Microtubule Dysfunction: A Common Feature of Neurodegenerative Diseases.

Neurons are particularly susceptible to microtubule (MT) defects and deregulation of the MT cytoskeleton is considered to be a common insult during the pathogenesis of neurodegenerative disorders. Evidence that dysfunctions in the MT system have a direct role in neurodegeneration comes from findings that several forms of neurodegenerative diseases are associated with changes in genes encoding tubulins, the structural units of MTs, MT-associated proteins (MAPs), or additional factors such as MT modifying enzymes which modulating tubulin post-translational modifications (PTMs) regulate MT functions and dynamics. Efforts to use MT-targeting therapeutic agents for the treatment of neurodegenerative diseases are underway. Many of these agents have provided several benefits when tested on both in vitro and in vivo neurodegenerative model systems. Currently, the most frequently addressed therapeutic interventions include drugs that modulate MT stability or that target tubulin PTMs, such as tubulin acetylation. The purpose of this review is to provide an update on the relevance of MT dysfunctions to the process of neurodegeneration and briefly discuss advances in the use of MT-targeting drugs for the treatment of neurodegenerative disorders.

TUBB Variants Underlying Different Phenotypes Result in Altered Vesicle Trafficking and Microtubule Dynamics.

Tubulinopathies are rare neurological disorders caused by alterations in tubulin structure and function, giving rise to a wide range of brain abnormalities involving neuronal proliferation, migration, differentiation and axon guidance. TUBB is one of the ten ß-tubulin encoding genes present in the human genome and is broadly expressed in the developing central nervous system and the skin. Mutations in TUBB are responsible for two distinct pathological conditions: the first is characterized by microcephaly and complex structural brain malformations and the second, also known as "circumferential skin creases Kunze type" (CSC-KT), is associated to neurological features, excess skin folding and growth retardation. We used a combination of immunocytochemical and cellular approaches to explore, on patients' derived fibroblasts, the functional consequences of two TUBB variants: the novel mutation (p.N52S), associated with basal ganglia and cerebellar dysgenesis, and the previously reported variant (p.M73T), linked to microcephaly, corpus callosum agenesis and CSC-KT skin phenotype. Our results demonstrate that these variants impair microtubule (MT) function and dynamics. Most importantly, our studies show an altered epidermal growth factor (EGF) and transferrin (Tf) intracellular vesicle trafficking in both patients' fibroblasts, suggesting a specific role of TUBB in MT-dependent vesicular transport.

Defective kinesin binding of TUBB2A causes progressive spastic ataxia syndrome resembling sacsinopathy.

Microtubules participate in fundamental cellular processes, including chromosomal segregation and cell division, migration and intracellular trafficking. Their proper function is required for correct central nervous system development and operative preservation, and mutations in genes coding tubulins, the constituting units of microtubules, underlie a family of neurodevelopmental and neurodegenerative diseases, collectively known as 'tubulinopathies', characterized by a wide range of neuronal defects resulting from defective proliferation, migration and function. Here, we causally link a previously unreported missense mutation in TUBB2A (c.1249G>A, p.D417N), encoding one of the neuron-specific ß-tubulin isotype II, to a disorder characterized by progressive spastic paraplegia, peripheral sensory-motor polyneuropathy and ataxia. Asp417 is a highly conserved solvent-exposed residue at the site mediating binding of kinesin superfamily motors. Impaired binding to KIF1A, a neuron-specific kinesin required for transport of synaptic vesicle precursors of the disease-associated TUBB2A mutant, was predicted by structural analyses and confirmed experimentally in vitro. We show that overexpression of TUBB2AD417N disrupts the mitotic spindle bipolarity and morphology and affects the M phase entry and length. Differently from the TUBB2AN247K and TUBB2AA248V, two mutants previously identified to affect neurodevelopment, TUBB2AD417N retains the ability to assemble into microtubules. Consistent with the differential clinical and structural impact, TUBB2AA248V does not drastically affect TUBB2A binding to KIF1A, nor mitotic spindle bipolarity. Overall, our data demonstrate a pathogenic role of the p.D417N substitution that is different from previously reported TUBB2A mutations and expand the phenotypic spectrum associated with mutations in this gene.

Cytoskeletal dynamics during in vitro neurogenesis of induced pluripotent stem cells (iPSCs).

Patient-derived induced pluripotent stem cells (iPSCs) provide a novel tool to investigate the pathophysiology of poorly known diseases, in particular those affecting the nervous system, which has been difficult to study for its lack of accessibility. In this emerging and promising field, recent iPSCs studies are mostly used as "proof-of-principle" experiments that are confirmatory of previous findings obtained from animal models and postmortem human studies; its promise as a discovery tool is just beginning to be realized. A recent number of studies point to the functional similarities between in vitro neurogenesis and in vivo neuronal development, suggesting that similar morphogenetic and patterning events direct neuronal differentiation. In this context, neuronal adhesion, cytoskeletal organization and cell metabolism emerge as an integrated and unexplored processes of human neurogenesis, mediated by the lack of data due to the difficult accessibility of the human neural tissue. These observations raise the necessity to understand which are the players controlling cytoskeletal reorganization and remodeling. In particular, we investigated human in vitro neurogenesis using iPSCs of healthy subjects to unveil the underpinnings of the cytoskeletal dynamics with the aim to shed light on the physiologic events controlling the development and the functionality of neuronal cells. We validate the iPSCs system to better understand the development of the human nervous system in order to set the bases for the future understanding of pathologies including developmental disorders (i.e. intellectual disability), epilepsy but also neurodegenerative disorders (i.e. Friedreich's Ataxia). We investigate the changes of the cytoskeletal components during the 30days of neuronal differentiation and we demonstrate that human neuronal differentiation requires a (time-dependent) reorganization of actin filaments, intermediate filaments and microtubules; and that immature neurons present a finely regulated localization of Glu-, Tyr- and Acet-TUBULINS. This study advances our understanding on cytoskeletal dynamics with the hope to pave the way for future therapies that could be potentially able to target cytoskeletal based neurodevelopmental and neurodegenerative diseases.

TBCE Mutations Cause Early-Onset Progressive Encephalopathy with Distal Spinal Muscular Atrophy.

Tubulinopathies constitute a family of neurodevelopmental/neurodegenerative disorders caused by mutations in several genes encoding tubulin isoforms. Loss-of-function mutations in TBCE, encoding one of the five tubulin-specific chaperones involved in tubulin folding and polymerization, cause two rare neurodevelopmental syndromes, hypoparathyroidism-retardation-dysmorphism and Kenny-Caffey syndrome. Although a missense mutation in Tbce has been associated with progressive distal motor neuronopathy in the pmn/pmn mice, no similar degenerative phenotype has been recognized in humans. We report on the identification of an early-onset and progressive neurodegenerative encephalopathy with distal spinal muscular atrophy resembling the phenotype of pmn/pmn mice and caused by biallelic TBCE mutations, with the c.464T>A (p.Ile155Asn) change occurring at the heterozygous/homozygous state in six affected subjects from four unrelated families originated from the same geographical area in Southern Italy. Western blot analysis of patient fibroblasts documented a reduced amount of TBCE, suggestive of rapid degradation of the mutant protein, similarly to what was observed in pmn/pmn fibroblasts. The impact of TBCE mutations on microtubule polymerization was determined using biochemical fractionation and analyzing the nucleation and growth of microtubules at the centrosome and extracentrosomal sites after treatment with nocodazole. Primary fibroblasts obtained from affected subjects displayed a reduced level of polymerized α-tubulin, similarly to tail fibroblasts of pmn/pmn mice. Moreover, markedly delayed microtubule re-polymerization and abnormal mitotic spindles with disorganized microtubule arrangement were also documented. Although loss of function of TBCE has been documented to impact multiple developmental processes, the present findings provide evidence that hypomorphic TBCE mutations primarily drive neurodegeneration.

Biallelic Mutations in TBCD, Encoding the Tubulin Folding Cofactor D, Perturb Microtubule Dynamics and Cause Early-Onset Encephalopathy.

Microtubules are dynamic cytoskeletal elements coordinating and supporting a variety of neuronal processes, including cell division, migration, polarity, intracellular trafficking, and signal transduction. Mutations in genes encoding tubulins and microtubule-associated proteins are known to cause neurodevelopmental and neurodegenerative disorders. Growing evidence suggests that altered microtubule dynamics may also underlie or contribute to neurodevelopmental disorders and neurodegeneration. We report that biallelic mutations in TBCD, encoding one of the five co-chaperones required for assembly and disassembly of the αß-tubulin heterodimer, the structural unit of microtubules, cause a disease with neurodevelopmental and neurodegenerative features characterized by early-onset cortical atrophy, secondary hypomyelination, microcephaly, thin corpus callosum, developmental delay, intellectual disability, seizures, optic atrophy, and spastic quadriplegia. Molecular dynamics simulations predicted long-range and/or local structural perturbations associated with the disease-causing mutations. Biochemical analyses documented variably reduced levels of TBCD, indicating relative instability of mutant proteins, and defective ß-tubulin binding in a subset of the tested mutants. Reduced or defective TBCD function resulted in decreased soluble α/ß-tubulin levels and accelerated microtubule polymerization in fibroblasts from affected subjects, demonstrating an overall shift toward a more rapidly growing and stable microtubule population. These cells displayed an aberrant mitotic spindle with disorganized, tangle-shaped microtubules and reduced aster formation, which however did not alter appreciably the rate of cell proliferation. Our findings establish that defective TBCD function underlies a recognizable encephalopathy and drives accelerated microtubule polymerization and enhanced microtubule stability, underscoring an additional cause of altered microtubule dynamics with impact on neuronal function and survival in the developing brain.

The cytoskeletal arrangements necessary to neurogenesis.

During the process of neurogenesis, the stem cell committed to the neuronal cell fate starts a series of molecular and morphological changes. The understanding of the physio-pathology of mechanisms controlling the molecular and morphological changes occurring during neuronal differentiation is fundamental to the development of effective therapies for many neurologic diseases. Unfortunately, our knowledge of the biological events occurring in the cell during neuronal differentiation is still poor. In this study, we focus preliminarily on the relevance of the cytoskeletal rearrangements, which earlier drive the morphology of the neuronal precursors, and later the migrating/mature neurons. In fact, neuritogenesis, neurite branching, outgrowth and retraction are seminal to the development of a fully functional nervous system. With this in mind, we highlight the importance of iPSC technology to study the processes of cytoskeletal-driven morphological changes during neuronal differentiation.

The interaction with HMG20a/b proteins suggests a potential role for beta-dystrobrevin in neuronal differentiation.

alpha and beta dystrobrevins are cytoplasmic components of the dystrophin-associated protein complex that are thought to play a role as scaffold proteins in signal transduction and intracellular transport. In the search of new insights into the functions of beta-dystrobrevin, the isoform restricted to non-muscle tissues, we performed a two-hybrid screen of a mouse cDNA library to look for interacting proteins. Among the positive clones, one encodes iBRAF/HMG20a, a high mobility group (HMG)-domain protein that activates REST (RE-1 silencing transcription factor)-responsive genes, playing a key role in the initiation of neuronal differentiation. We characterized the beta-dystrobrevin-iBRAF interaction by in vitro and in vivo association assays, localized the binding region of one protein to the other, and assessed the kinetics of the interaction as one of high affinity. We also found that beta-dystrobrevin directly binds to BRAF35/HMG20b, a close homologue of iBRAF and a member of a co-repressor complex required for the repression of neural specific genes in neuronal progenitors. In vitro assays indicated that beta-dystrobrevin binds to RE-1 and represses the promoter activity of synapsin I, a REST-responsive gene that is a marker for neuronal differentiation. Altogether, our data demonstrate a direct interaction of beta-dystrobrevin with the HMG20 proteins iBRAF and BRAF35 and suggest that beta-dystrobrevin may be involved in regulating chromatin dynamics, possibly playing a role in neuronal differentiation.