Recent advances in the genetics of distal hereditary motor neuropathy give insight to a disease mechanism involving copper homeostasis that may extend to other motor neuron disorders

Distal hereditary motor neuropathy (dHMN) is a sub-group of Charcot-Marie-Tooth disease (CMT), the most common peripheral neuropathy, that affects only motor neurons. The recent observation of ATP7A mutations in dHMN provides insight for a common disease mechanism that may involve copper homeostasis. Functionally, diverse proteins were previously shown to underlie dHMN and a convergent link is destined to unfold for some of these. We propose connections between copper and known dHMN genes that overlap also with the causative genes of other motor neuron disorders (MNDs).     

An Overview and Update of ATP7A Mutations Leading to Menkes Disease and Occipital Horn Syndrome

Menkes disease (MD) is a lethal multisystemic disorder of copper metabolism. Progressive neurodegeneration and connective tissue disturbances, together with the peculiar “kinky” hair, are the main manifestations. MD is inherited as an X‐linked recessive trait, and as expected the vast majority of patients are males. MD occurs because of mutations in the ATP7A gene and the vast majority of ATP7A mutations are intragenic mutations or partial gene deletions. ATP7A is an energy‐dependent transmembrane protein, which is involved in the delivery of copper to the secreted copper enzymes and in the export of surplus copper from cells. Severely affected MD patients die usually before the third year of life. A cure for the disease does not exist, but very early copper–histidine treatment may correct some of the neurological symptoms. This study reviews 274 published and 18 novel disease causing mutations identified in 370 unrelated MD patients, nonpathogenic variants of ATP7A, functional studies of the ATP7A mutations, and animal models of MD.     

Extension of the phenotype of biallelic loss‐of‐function mutations in SLC25A46 to the severe form of pontocerebellar hypoplasia type I

Biallelic mutations in SLC25A46, encoding a modified solute transporter involved in mitochondrial dynamics, have been identified in a wide range of conditions such as hereditary motor and sensory neuropathy with optic atrophy type VIB (OMIM: *610826) and congenital lethal pontocerebellar hypoplasia (PCH). To date, 18 patients from 13 families have been reported, presenting with the key clinical features of optic atrophy, peripheral neuropathy, and cerebellar atrophy. The course of the disease was highly variable ranging from severe muscular hypotonia at birth and early death to first manifestations in late childhood and survival into the fifties. Here we report on 4 patients from 2 families diagnosed with PCH who died within the first month of life from respiratory insufficiency. Patients from 1 family had pathoanatomically proven spinal motor neuron degeneration (PCH1). Using exome sequencing, we identified biallelic disease‐segregating loss‐of‐function mutations in SLC25A46 in both families. Our study adds to the definition of the SLC25A46‐associated phenotypic spectrum that includes neonatal fatalities due to PCH as the severe extreme.     

Biallelic variant in AGTPBP1 causes infantile lower motor neuron degeneration and cerebellar atrophy

Infantile hereditary lower motor neuron disorders beyond 5q–spinal muscular atrophy (5q‐SMA) are usually caused by mutations other than deletions or mutations in SMN1. In addition to motor neuron degeneration, further neurologic or multisystemic pathologies in non‐5q‐SMAs are not seldom. Some of the non‐5q‐SMA phenotypes, such as pontocerebellar hypoplasia (PCH1), have been classified later as a different disease group due to distinctive primary pathologies. Likewise, a novel phenotype, childhood‐onset neurodegeneration with cerebellar atrophy (CONDCA) has been described recently in individuals with lower motor neuron disorder and cerebellar atrophy due to biallelic loss‐of‐function variants in AGTPBP1 that encodes cytosolic carboxypeptidase 1 (CCP1). Here we present two individuals with CONDCA in whom a biallelic missense AGTPBP1 variant (NM_001330701.1:c.2396G>T, p.Arg799Leu) was identified by whole exome sequencing. Affected individuals in this report correspond to the severe infantile spectrum of the disease and underline the severe pathogenic effect of this missense variant. This report is the second in the literature that delineates the pathogenic effects of biallelic AGTPBP1 variants presenting the recently described CONDCA disease.     

Favorably skewed X‐inactivation accounts for neurological sparing in female carriers of Menkes disease

Desai V, Donsante A, Swoboda KJ, Martensen M, Thompson J, Kaler SG. Favorably skewed X‐inactivation accounts for neurological sparing in female carriers of Menkes disease. Classical Menkes disease is an X‐linked recessive neurodegenerative disorder caused by mutations in ATP7A, which is located at Xq13.1‐q21. ATP7A encodes a copper‐transporting P‐type ATPase and plays a critical role in development of the central nervous system. With rare exceptions involving sex chromosome aneuploidy or X‐autosome translocations, female carriers of ATP7A mutations are asymptomatic except for subtle hair and skin abnormalities, although the mechanism for this neurological sparing has not been reported. We studied a three‐generation family in which a severe ATP7A mutation, a 5.5‐kb genomic deletion spanning exons 13 and 14, segregated. The deletion junction fragment was amplified from the proband by long‐range polymerase chain reaction and sequenced to characterize the breakpoints. We screened at‐risk females in the family for this junction fragment and analyzed their X‐inactivation patterns using the human androgen‐receptor (HUMARA) gene methylation assay. We detected the junction fragment in the proband, two obligate heterozygotes, and four of six at‐risk females. Skewed inactivation of the X chromosome harboring the deletion was noted in all female carriers of the deletion (n = 6), whereas random X‐inactivation was observed in all non‐carriers (n = 2). Our results formally document one mechanism for neurological sparing in female carriers of ATP7A mutations. Based on review of X‐inactivation patterns in female carriers of other X‐linked recessive diseases, our findings imply that substantial expression of a mutant ATP7A at the expense of the normal allele could be associated with neurologic symptoms in female carriers of Menkes disease and its allelic variants, occipital horn syndrome, and ATP7A‐related distal motor neuropathy.     

Selective loss of alpha motor neurons with sparing of gamma motor neurons and spinal cord cholinergic neurons in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neuromuscular disease characterised primarily by loss of lower motor neurons from the ventral grey horn of the spinal cord and proximal muscle atrophy. Recent experiments utilising mouse models of SMA have demonstrated that not all motor neurons are equally susceptible to the disease, revealing that other populations of neurons can also be affected. Here, we have extended investigations of selective vulnerability of neuronal populations in the spinal cord of SMA mice to include comparative assessments of alpha motor neuron (α‐MN) and gamma motor neuron (γ‐MN) pools, as well as other populations of cholinergic neurons. Immunohistochemical analyses of late‐symptomatic SMA mouse spinal cord revealed that numbers of α‐MNs were significantly reduced at all levels of the spinal cord compared with controls, whereas numbers of γ‐MNs remained stable. Likewise, the average size of α‐MN cell somata was decreased in SMA mice with no change occurring in γ‐MNs. Evaluation of other pools of spinal cord cholinergic neurons revealed that pre‐ganglionic sympathetic neurons, central canal cluster interneurons, partition interneurons and preganglionic autonomic dorsal commissural nucleus neuron numbers all remained unaffected in SMA mice. Taken together, these findings indicate that α‐MNs are uniquely vulnerable among cholinergic neuron populations in the SMA mouse spinal cord, with γ‐MNs and other cholinergic neuronal populations being largely spared.     

Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy

ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7A(T994I) and ATP7A(P1386S), with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7A(T994I) and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7A(T994I) mislocalization. Flow cytometry documented that non-permeabilized ATP7A(P1386S) fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.     

Sensory neuropathy in progressive motor neuronopathy (pmn) mice is associated with defects in microtubule polymerization and axonal transport

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) are now recognized as multi‐system disorders also involving various non‐motor neuronal cell types. The precise extent and mechanistic basis of non‐motor neuron damage in human ALS and ALS animal models remain however unclear. To address this, we here studied progressive motor neuronopathy (pmn) mice carrying a missense loss‐of‐function mutation in tubulin binding cofactor E (TBCE). These mice manifest a particularly aggressive form of motor axon dying back and display a microtubule loss, similar to that induced by human ALS‐linked TUBA4A mutations. Using whole nerve confocal imaging of pmn × thy1.2‐YFP16 fluorescent reporter mice and electron microscopy, we demonstrate axonal discontinuities, bead‐like spheroids and ovoids in pmn suralis nerves indicating prominent sensory neuropathy. The axonal alterations qualitatively resemble those in phrenic motor nerves but do not culminate in the loss of myelinated fibers. We further show that the pmn mutation decreases the level of TBCE, impedes microtubule polymerization in dorsal root ganglion (DRG) neurons and causes progressive loss of microtubules in large and small caliber suralis axons. Live imaging of axonal transport using GFP‐tagged tetanus toxin C‐fragment (GFP‐TTC) demonstrates defects in microtubule‐based transport in pmn DRG neurons, providing a potential explanation for the axonal alterations in sensory nerves. This study unravels sensory neuropathy as a pathological feature of mouse pmn, and discusses the potential contribution of cytoskeletal defects to sensory neuropathy in human motor neuron disease.     

Overexpression of survival motor neuron improves neuromuscular function and motor neuron survival in mutant SOD1 mice

Spinal muscular atrophy results from diminished levels of survival motor neuron (SMN) protein in spinal motor neurons. Low levels of SMN also occur in models of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1) and genetic reduction of SMN levels exacerbates the phenotype of transgenic SOD1^G93A mice. Here, we demonstrate that SMN protein is significantly reduced in the spinal cords of patients with sporadic ALS. To test the potential of SMN as a modifier of ALS, we overexpressed SMN in 2 different strains of SOD1^G93A mice. Neuronal overexpression of SMN significantly preserved locomotor function, rescued motor neurons, and attenuated astrogliosis in spinal cords of SOD1^G93A mice. Despite this, survival was not prolonged, most likely resulting from SMN mislocalization and depletion of gems in motor neurons of symptomatic mice. Our results reveal that SMN upregulation slows locomotor deficit onset and motor neuron loss in this mouse model of ALS. However, disruption of SMN nuclear complexes by high levels of mutant SOD1, even in the presence of SMN overexpression, might limit its survival promoting effects in this specific mouse model. Studies in emerging mouse models of ALS are therefore warranted to further explore the potential of SMN as a modifier of ALS.     

KIF5A de novo mutation associated with myoclonic seizures and neonatal onset progressive leukoencephalopathy

The KIF5A gene (OMIM 602821) encodes a neuron‐specific kinesin heavy chain involved in intracellular transport of mitochondria and other cargoes. KIF5A protein comprises the N terminal motor domain, the stalk domain and the C‐terminal cargo binding domain. The binding between KIF5A and its cargoes is mediated by kinesin adaptor proteins such as TRAK1 and TRAK2. Numerous missense KIF5A mutations in the motor and stalk domains cause spastic paraplegia type 10 (SPG10, OMIM 604187). Conversely, the role of loss‐of‐function mutations, especially those affecting the cargo binding domain, is unclear. We describe a novel de novo KIF5A p.Ser974fs/c.2921delC mutation found by whole exome sequencing in a patient with a congenital severe disease characterized by myoclonic seizures and progressive leukoencephalopathy. Since this phenotype differs considerably from the KIF5A/SPG10 disease spectrum we propose that the KIF5A p.Ser974fs and possibly other mutations which lead to truncation of the C‐terminal tail of the protein cause a novel disorder. We speculate that the unique effect of the C‐terminal truncating KIF5A mutations may result from the previously described complex role of this protein domain in binding of the TRAK2 and possibly other kinesin adaptor protein(s).     

A 37‐year‐old Menkes disease patient—Residual ATP7A activity and early copper administration as key factors in beneficial treatment

Menkes disease (MD) is a lethal disorder characterized by severe neurological symptoms and connective tissue abnormalities; and results from malfunctioning of cuproenzymes, which cannot receive copper due to a defective intracellular copper transporting protein, ATP7A. Early parenteral copper‐histidine supplementation may modify disease progression substantially but beneficial effects of long‐term treatment have been recorded in only a few patients. Here we report on the eldest surviving MD patient (37 years) receiving early‐onset and long‐term copper treatment. He has few neurological symptoms without connective tissue disturbances; and a missense ATP7A variant, p.(Pro852Leu), which results in impaired protein trafficking while the copper transport function is spared. These findings suggest that some cuproenzymes maintain their function when sufficient copper is provided to the cells; and underline the importance of early initiated copper treatment, efficiency of which is likely to be dependent on the mutant ATP7A function.     

Spinal Muscular Atrophy: New Findings for an Old Pathology

Understanding the events that are responsible for a disease is mandatory for setting up a therapeutic strategy. Although spinal muscular atrophy (SMA) is considered a rare neurodegenerative pathology, its impact in our society is really devastating as it strikes young people from birth onward, and it affects their families either emotionally or financially. Moreover, it requires intensive care for the children, and this diverts both parents and relatives from their occupations. Each neuron is very different from one another; therefore, in a neurodegenerative disease, the population of axons, synapses and cell bodies degenerate asynchronously, and subpopulations of neurons have different vulnerabilities. The knowledge of the sequence of events along the lengths of individual neurons is crucial to understand if each synapse degenerates before the corresponding axon, or if each axon degenerates before the corresponding cell body. Early degeneration of one neuronal compartment in disease often reflects molecular defects somewhere else. Up until now, SMA is considered mostly a lower motor neuron disease caused by the loss‐of‐function mutations in the SMN1 gene; here, we inspect other features that can be altered by this defect, such as the cross talk between muscle and motor neuron and the role of physical inactivity.     

Extracellular ATP and the P2X<Subscript>7</Subscript>receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

Background  

During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X₇ receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1^G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X₇ receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1^G93A astrocytes.     

Perspectives and diagnostic considerations in spinal muscular atrophy

Spinal muscular atrophy is an autosomal recessive neurodegenerative disease and the most common genetic cause of infant mortality. The disease results in motor neuron loss and skeletal muscle atrophy. Spinal muscular atrophy is caused by mutations in the telomeric copy of the survival motor neuron 1 (SMN1) gene, but all patients retain a centromeric copy of the gene, SMN2. In the majority of cases, the disease severity correlates inversely with an increased SMN2 gene copy number. Because spinal muscular atrophy is both a severe and common disorder, a direct carrier testing has been beneficial to many families. The survival motor neuron protein is ubiquitously expressed and performs a role in the assembly of the spliceosome. It is still not understood why mutations in the SMN1 gene only seem to affect motor neurons. Progress has been made by developing therapeutic strategies based on understanding the pathogenesis of the disease. This review attempts to highlight some of the recent advances in the understanding of the disease with a focus on molecular diagnostics.     

De Novo Mutations in the Motor Domain of KIF1A Cause Cognitive Impairment,Spastic Paraparesis,Axonal Neuropathy,and Cerebellar Atrophy

KIF1A is a neuron‐specific motor protein that plays important roles in cargo transport along neurites. Recessive mutations in KIF1A were previously described in families with spastic paraparesis or sensory and autonomic neuropathy type‐2. Here, we report 11 heterozygous de novo missense mutations (p.S58L, p.T99M, p.G102D, p.V144F, p.R167C, p.A202P, p.S215R, p.R216P, p.L249Q, p.E253K, and p.R316W) in KIF1A in 14 individuals, including two monozygotic twins. Two mutations (p.T99M and p.E253K) were recurrent, each being found in unrelated cases. All these de novo mutations are located in the motor domain (MD) of KIF1A. Structural modeling revealed that they alter conserved residues that are critical for the structure and function of the MD. Transfection studies suggested that at least five of these mutations affect the transport of the MD along axons. Individuals with de novo mutations in KIF1A display a phenotype characterized by cognitive impairment and variable presence of cerebellar atrophy, spastic paraparesis, optic nerve atrophy, peripheral neuropathy, and epilepsy. Our findings thus indicate that de novo missense mutations in the MD of KIF1A cause a phenotype that overlaps with, while being more severe, than that associated with recessive mutations in the same gene.     

A homozygous canonical splice acceptor site mutation in PRUNE1 is responsible for a rare childhood neurodegenerative disease in Manitoba Cree families

Autosomal recessive PRUNE1 mutations are reported to cause a severe neurodevelopmental disorder with microcephaly, hypotonia, and brain malformations. We describe clinical and neuropathological features in a cohort of nine individuals of Cree descent who, because of a founder effect, are homozygous for the same PRUNE1 mutation. They follow the course of a combined neuromuscular and neurodegenerative disease, rather than a pure failure of normal development. This cohort presented in infancy with features of lower motor neuron disease, such as hypotonia, contractures, tongue fasciculations, and feeding difficulties in the absence of congenital brain anomalies and microcephaly. A neurodegenerative course followed with onset of seizures, spasticity, and respiratory insufficiency. Muscle biopsies showed denervation/reinnervation features, nonspecific atrophy and end‐stage atrophy. Autopsy findings in two patients are also described, suggesting length dependent central motor axon degeneration, peripheral motor axon degeneration, possible spinal motor neuron degeneration, and accumulation of beta amyloid precursor protein inclusions in select brainstem nuclei. Exome sequencing and homozygosity mapping identified a homozygous PRUNE1 mutation in a canonical splice site, which produces two abnormal PRUNE1 mRNA products. Based on our studies and the histopathology and phenotypic data, we provide further evidence that this disorder leads to a neurodegenerative disease affecting both the peripheral and central nervous systems and suggest that the pathogenic c.521‐2A>G mutation could lead to an altered effect on tubulin dynamics.     

Characterization of Ighmbp2 in motor neurons and implications for the pathomechanism in a mouse model of human spinal muscular atrophy with respiratory distress type 1 (SMARD1)

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is caused by recessive mutations of the IGHMBP2 gene. The role of IGHMBP2 (immunoglobulin mu-binding protein 2) in the pathomechanism of motor neuron disease is unknown. We have generated antibodies against Ighmbp2 and showed that low levels of Ighmbp2 immunoreactivity are present in the nucleus of spinal motor neurons and high levels in cell bodies, axons and growth cones. Ighmbp2 protein levels are strongly reduced in neuromuscular degeneration (nmd) mice, the mouse model of SMARD1. Mutant mice show severe motor neuron degeneration before first clinical symptoms become apparent. The loss of motor neuron cell bodies in lumbar spinal cord is followed by axonal degeneration in corresponding nerves such as the femoral quadriceps and sciatic nerve and loss of axon terminals at motor endplates. Motor neuron degeneration and clinical symptoms then slowly progress until the mice die at the age of 3-4 months. In addition, myopathic changes seem to contribute to muscle weakness and especially to respiratory failure, which is characteristic of the disorder in humans. Cultured motor neurons from embryonic nmd mice did not show any abnormality with respect to survival, axonal growth or growth cone size, thus differing from motor neurons derived from, e.g. Smn (survival motor neuron) deficient mice, the model of spinal muscular atrophy (SMA). Our data suggest that the pathomechanism in SMARD1 is clearly distinct from other motor neuron diseases such as classic SMA.     

ATP1A1 mutations cause intermediate Charcot‐Marie‐Tooth disease

Intermediate Charcot‐Marie‐Tooth (CMT) disease is a heterogeneous group of inherited neuropathies characterized by progressive muscle weakness and atrophy of the distal extremities, distal sensory loss. There were still a large proportion of causative genes for intermediate CMT failed to be identified. Here, using whole‐exome sequencing technique, we identified two novel missense mutations in ATP1A1 gene, c.620C>T (p.S207F) and c.2629G>A (p.G877S), in two Chinese CMT families. Further functional analysis revealed that these mutations led to the loss function of the ATP1A1 protein. The two mutations did not affect the levels of messenger RNA but possessed a damaging effect on ATP1A1 protein expression and they downregulated the protein levels of ATP1A1 by promoting its proteasome degradation. Taken together, we confirmed ATP1A1 as a novel causative gene for intermediate CMT.     

Du gène à la maladie : les anomalies des transporteurs du cuivre

Copper is a trace metal, essential for many biological processes. Copper is also toxic if in excessive amounts; its homeostatic balance requires a delicate regulation. Several severe hereditary human disorders of copper regulatory mechanisms have been identified; they are related to mutations in gene ATP7A and ATP7B coding for copper-transporting proteins. Those mutations result in copper deficiency for ATP7A (Menkes disease) and copper overload for ATP7B (Wilson disease). Usually, clinical and biochemical phenotypes of these diseases are disparate. This article focuses on the molecular pathogenesis of Wilson and Menkes disease, and discusses how causing mutations are correlated with molecular defects and disease phenotypes.