Alexander J. Abrams

Treatable childhood neuronopathy caused by mutations in riboflavin transporter RFVT2

Childhood onset motor neuron diseases or neuronopathies are a clinically heterogeneous group of disorders. A particularly severe subgroup first described in 1894, and subsequently called Brown-Vialetto-Van Laere syndrome, is characterized by progressive pontobulbar palsy, sensorineural hearing loss and respiratory insufficiency. There has been no treatment for this progressive neurodegenerative disorder, which leads to respiratory failure and usually death during childhood. We recently reported the identification of SLC52A2, encoding riboflavin transporter RFVT2, as a new causative gene for Brown-Vialetto-Van Laere syndrome. We used both exome and Sanger sequencing to identify SLC52A2 mutations in patients presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. We undertook clinical, neurophysiological and biochemical characterization of patients with mutations in SLC52A2, functionally analysed the most prevalent mutations and initiated a regimen of high-dose oral riboflavin. We identified 18 patients from 13 families with compound heterozygous or homozygous mutations in SLC52A2. Affected individuals share a core phenotype of rapidly progressive axonal sensorimotor neuropathy (manifesting with sensory ataxia, severe weakness of the upper limbs and axial muscles with distinctly preserved strength of the lower limbs), hearing loss, optic atrophy and respiratory insufficiency. We demonstrate that SLC52A2 mutations cause reduced riboflavin uptake and reduced riboflavin transporter protein expression, and we report the response to high-dose oral riboflavin therapy in patients with SLC52A2 mutations, including significant and sustained clinical and biochemical improvements in two patients and preliminary clinical response data in 13 patients with associated biochemical improvements in 10 patients. The clinical and biochemical responses of this SLC52A2-specific cohort suggest that riboflavin supplementation can ameliorate the progression of this neurodegenerative condition, particularly when initiated soon after the onset of symptoms.

Mutations in SLC25A46, encoding a UGO1-like protein, cause an optic atrophy spectrum disorder

Dominant optic atrophy (DOA) and axonal peripheral neuropathy (Charcot-Marie-Tooth type 2, or CMT2) are hereditary neurodegenerative disorders most commonly caused by mutations in the canonical mitochondrial fusion genes OPA1 and MFN2, respectively. In yeast, homologs of OPA1 (Mgm1) and MFN2 (Fzo1) work in concert with Ugo1, for which no human equivalent has been identified thus far. By whole-exome sequencing of patients with optic atrophy and CMT2, we identified four families with recessive mutations in SLC25A46. We demonstrate that SLC25A46, like Ugo1, is a modified carrier protein that has been recruited to the outer mitochondrial membrane and interacts with the inner membrane remodeling protein mitofilin (Fcj1). Loss of function in cultured cells and in zebrafish unexpectedly leads to increased mitochondrial connectivity, while severely affecting the development and maintenance of neurons in the fish. The discovery of SLC25A46 strengthens the genetic overlap between optic atrophy and CMT2 while exemplifying a new class of modified solute transporters linked to mitochondrial dynamics.

The Expanding Role of MBD Genes in Autism: Identification of a MECP2 Duplication and Novel Alterations in MBD5, MBD6, and SETDB1

The methyl‐CpG‐binding domain (MBD) gene family was first linked to autism over a decade ago when Rett syndrome, which falls under the umbrella of autism spectrum disorders (ASDs), was revealed to be predominantly caused by MECP2 mutations. Since that time, MECP2 alterations have been recognized in idiopathic ASD patients by us and others. Individuals with deletions across the MBD5 gene also present with ASDs, impaired speech, intellectual difficulties, repetitive behaviors, and epilepsy. These findings suggest that further investigations of the MBD gene family may reveal additional associations related to autism. We now describe the first study evaluating individuals with ASD for rare variants in four autosomal MBD family members, MBD5, MBD6, SETDB1, and SETDB2, and expand our initial screening in the MECP2 gene. Each gene was sequenced over all coding exons and evaluated for copy number variations in 287 patients with ASD and an equal number of ethnically matched control individuals. We identified 186 alterations through sequencing, approximately half of which were novel (96 variants, 51.6%). We identified 17 ASD specific, nonsynonymous variants, four of which were concordant in multiplex families: MBD5 Tyr1269Cys, MBD6 Arg883Trp, MECP2 Thr240Ser, and SETDB1 Pro1067del. Furthermore, a complex duplication spanning of the MECP2 gene was identified in two brothers who presented with developmental delay and intellectual disability. From our studies, we provide the first examples of autistic patients carrying potentially detrimental alterations in MBD6 and SETDB1, thereby demonstrating that the MBD gene family potentially plays a significant role in rare and private genetic causes of autism. Autism Res 2012, 5: 385–397.

Transgenic complementation of MeCP2 deficiency: phenotypic rescue of Mecp2-null mice by isoform-specific transgenes

Rett syndrome (RTT) is a disorder that affects patients’ ability to communicate, move and behave. RTT patients are characterized by impaired language, stereotypic behaviors, frequent seizures, ataxia and sleep disturbances, with the onset of symptoms occurring after a period of seemingly normal development. RTT is caused by mutations in methyl-CpG binding protein 2 (MECP2), an X-chromosome gene encoding for MeCP2, a protein that regulates gene expression. MECP2 generates two alternative splice variants encoding two protein isoforms that differ only in the N-terminus. Although no functional differences have been identified for these splice variants, it has been suggested that the RTT phenotype may occur in the presence of a functional MeCP2-e2 protein. This suggests that the two isoforms might be functionally distinct. Supporting this notion, the two variants show regional and age-related differences in transcript abundance. Here, we show that transgenic expression of either the MeCP2-e1 or MeCP2-e2 splice variant results in prevention of development of RTT-like phenotypic manifestations in a mouse model lacking Mecp2. Our results indicate that the two MeCP2 splice variants can substitute for each other and fulfill the basic functions of MeCP2 in the mouse brain.

S-Nitrosylation of ApoE in Alzheimer’s Disease

The mechanism by which apolipoprotein E (ApoE) isoforms functionally influence the risk and progression of late-onset Alzheimers disease (LOAD) remains hitherto unknown. Herein, we present evidence that all ApoE isoforms bind to nitric oxide synthase 1 (NOS1) and that such protein-protein interaction results in S-nitrosylation of ApoE2 and ApoE3 but not ApoE4. Our structural analysis at the atomic level reveals that S-nitrosylation of ApoE2 and ApoE3 proteins may lead to conformational changes resulting in the loss of binding to low-density lipoprotein (LDL) receptors. Collectively, our data suggest that S-nitrosylation of ApoE proteins may play an important role in regulating lipid metabolism and in the pathogenesis of LOAD.

Disruption of Mbd5 in mice causes neuronal functional deficits and neurobehavioral abnormalities consistent with 2q23.1 microdeletion syndrome

2q23.1 microdeletion syndrome is characterized by intellectual disability, motor delay, autistic‐like behaviors, and a distinctive craniofacial phenotype. All patients carry a partial or total deletion of methyl‐CpG‐binding domain protein 5 (MBD5), suggesting that haploinsufficiency of this gene is responsible for the phenotype. To confirm this hypothesis and to examine the role of MBD5 in vivo, we have generated and characterized an Mbd5 gene‐trap mouse model. Our study indicates that the Mbd5+/GT mouse model recapitulates most of the hallmark phenotypes observed in 2q23.1 deletion carriers including abnormal social behavior, cognitive impairment, and motor and craniofacial abnormalities. In addition, neuronal cultures uncovered a deficiency in neurite outgrowth. These findings support a causal role of MBD5 in 2q23.1 microdeletion syndrome and suggest a role for MBD5 in neuronal processes. The Mbd5+/GT mouse model will advance our understanding of the abnormal brain development underlying the emergence of 2q23.1 deletion‐associated behavioral and cognitive symptoms.

Function Over Form: Modeling Groups of Inherited Neurological Conditions in Zebrafish

Zebrafish are a unique cell to behavior model for studying the basic biology of human inherited neurological conditions. Conserved vertebrate genetics and optical transparency provide in vivo access to the developing nervous system as well as high-throughput approaches for drug screens. Here we review zebrafish modeling for two broad groups of inherited conditions that each share genetic and molecular pathways and overlap phenotypically: neurodevelopmental disorders such as Autism Spectrum Disorders (ASD), Intellectual Disability (ID) and Schizophrenia (SCZ), and neurodegenerative diseases, such as Cerebellar Ataxia (CATX), Hereditary Spastic Paraplegia (HSP) and Charcot-Marie Tooth Disease (CMT). We also conduct a small meta-analysis of zebrafish orthologs of high confidence neurodevelopmental disorder and neurodegenerative disease genes by looking at duplication rates and relative protein sizes. In the past zebrafish genetic models of these neurodevelopmental disorders and neurodegenerative diseases have provided insight into cellular, circuit and behavioral level mechanisms contributing to these conditions. Moving forward, advances in genetic manipulation, live imaging of neuronal activity and automated high-throughput molecular screening promise to help delineate the mechanistic relationships between different types of neurological conditions and accelerate discovery of therapeutic strategies.

Cryptic amyloidogenic elements in mutant NEFH causing Charcot-Marie-Tooth 2 trigger aggresome formation and neuronal death

Neurofilament heavy chain (NEFH) gene was recently identified to cause autosomal dominant axonal Charcot-Marie-Tooth disease (CMT2cc). However, the clinical spectrum of this condition and the physio-pathological pathway remain to be delineated. We report 12 patients from two French families with axonal dominantly inherited form of CMT caused by two new mutations in the NEFH gene. A remarkable feature was the early involvement of proximal muscles of the lower limbs associated with pyramidal signs in some patients. Nerve conduction velocity studies indicated a predominantly motor axonal neuropathy. Unique deletions of two nucleotides causing frameshifts near the end of the NEFH coding sequence were identified: in family 1, c.3008_3009del (p.Lys1003Argfs*59), and in family 2 c.3043_3044del (p.Lys1015Glyfs*47). Both frameshifts lead to 40 additional amino acids translation encoding a cryptic amyloidogenic element. Consistently, we show that these mutations cause protein aggregation which are recognised by the autophagic pathway in motoneurons and triggered caspase 3 activation leading to apoptosis in neuroblastoma cells. Using electroporation of chick embryo spinal cord, we confirm that NEFH mutants form aggregates in vivo and trigger apoptosis of spinal cord neurons. Thus, our results provide a physiological explanation for the overlap between CMT and amyotrophic lateral sclerosis (ALS) clinical features in affected patients.

Insights into the genotype-phenotype correlation and molecular function of SLC25A46

Recessive SLC25A46 mutations cause a spectrum of neurodegenerative disorders with optic atrophy as a core feature. We report a patient with optic atrophy, peripheral neuropathy, ataxia, but not cerebellar atrophy, who is on the mildest end of the phenotypic spectrum. By studying seven different nontruncating mutations, we found that the stability of the SLC25A46 protein inversely correlates with the severity of the disease and the patients variant does not markedly destabilize the protein. SLC25A46 belongs to the mitochondrial transporter family, but it is not known to have transport function. Apart from this possible function, SLC25A46 forms molecular complexes with proteins involved in mitochondrial dynamics and cristae remodeling. We demonstrate that the patients mutation directly affects the SLC25A46 interaction with MIC60. Furthermore, we mapped all of the reported substitutions in the protein onto a 3D model and found that half of them fall outside of the signature carrier motifs associated with transport function. We thus suggest that there are two distinct molecular mechanisms in SLC25A46‐associated pathogenesis, one that destabilizes the protein while the other alters the molecular interactions of the protein. These results have the potential to inform clinical prognosis of such patients and indicate a pathway to drug target development.