To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600).
Target exon capture and next generation sequencing was ...used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed.
Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state-a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function.
We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein (DYNC1H1) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance.
Mitochondrial dysfunction is a common cause of peripheral neuropathies. While the role of neuron and axonal mitochondria in peripheral nerve disease is well appreciated, whether Schwann cell (SC) ...mitochondrial deficits contribute to peripheral neuropathies is unclear. Here, we examine how SC mitochondrial dysfunction affects axonal survival and contributes to the decline of peripheral nerve function by generating mice with SC-specific mitochondrial deficits. These mice (Tfam-SCKOs) were produced through the tissue-specific deletion of the mitochondrial transcription factor A gene (Tfam), which is essential for mitochondrial DNA (mtDNA) transcription and maintenance. Tfam-SCKOs were viable, but as they aged, they developed a progressive peripheral neuropathy characterized by nerve conduction abnormalities as well as extensive muscle denervation. Morphological examination of Tfam-SCKO nerves revealed early preferential loss of small unmyelinated fibers followed by prominent demyelination and degeneration of larger-caliber axons. Tfam-SCKOs displayed sensory and motor deficits consistent with this pathology. Remarkably, the severe mtDNA depletion and respiratory chain abnormalities in Tfam-SCKO mice did not affect SC proliferation or survival. Mitochondrial function in SCs is therefore essential for maintenance of axonal survival and normal peripheral nerve function, suggesting that SC mitochondrial dysfunction contributes to human peripheral neuropathies.
Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. ...Decreased expression of C9orf72 is seen in expansion carriers, suggesting that loss of function may play a role in disease. We found that two independent mouse lines lacking the C9orf72 ortholog (3110043O21Rik) in all tissues developed normally and aged without motor neuron disease. Instead, C9orf72 null mice developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophage-like cells. C9orf72 expression was highest in myeloid cells, and the loss of C9orf72 led to lysosomal accumulation and altered immune responses in macrophages and microglia, with age-related neuroinflammation similar to C9orf72 ALS but not sporadic ALS human patient tissue. Thus, C9orf72 is required for the normal function of myeloid cells, and altered microglial function may contribute to neurodegeneration in C9orf72 expansion carriers.
The glial cell line derived neurotrophic factor (GDNF) family has recently been expanded to include four members, and the interactions between these neurotrophic factors and their unique receptor ...system is now beginning to be understood. Furthermore, analysis of mice lacking the genes for GDNF, neurturin, and their related receptors has confirmed the importance of these factors in neurodevelopment. The results of such analyses reveal numerous similarities and potential overlaps in the way the GDNF and the nerve growth factor (NGF) families regulate development of the peripheral nervous system.
Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the ...noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased, leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS.
Spinal muscular atrophies (SMAs) are hereditary disorders characterized by weakness from degeneration of spinal motor neurons. Although most SMA cases with proximal weakness are recessively ...inherited, rare families with dominant inheritance have been reported. We aimed to clinically, pathologically, and genetically characterize a large North American family with an autosomal dominant proximal SMA.
Affected family members underwent clinical and electrophysiologic evaluation. Twenty family members were genotyped on high-density genome-wide SNP arrays and linkage analysis was performed.
Ten affected individuals (ages 7-58 years) showed prominent quadriceps atrophy, moderate to severe weakness of quadriceps and hip abductors, and milder degrees of weakness in other leg muscles. Upper extremity strength and sensation was normal. Leg weakness was evident from early childhood and was static or very slowly progressive. Electrophysiology and muscle biopsies were consistent with chronic denervation. SNP-based linkage analysis showed a maximum 2-point lod score of 5.10 (theta = 0.00) at rs17679127 on 14q32. A disease-associated haplotype spanning from 114 cM to the 14q telomere was identified. A single recombination narrowed the minimal genomic interval to Chr14: 100,220,765-106,368,585. No segregating copy number variations were found within the disease interval.
We describe a family with an early onset, autosomal dominant, proximal SMA with a distinctive phenotype: symptoms are limited to the legs and there is notable selectivity for the quadriceps. We demonstrate linkage to a 6.1-Mb interval on 14q32 and propose calling this disorder spinal muscular atrophy-lower extremity, dominant.
This review examines the recent research developments aimed at defining the role of RNA-binding proteins (TDP-43 and FUS) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration ...(FTLD).
TAR DNA-binding protein 43 kDa (TDP-43) and fused in sarcoma (FUS) are RNA-binding proteins that form aggregates in ALS and FTLD, and when mutated can drive the pathogenesis of these disorders. However, fundamental questions remain as to the relationship between TDP-43 and FUS aggregation and disease, their normal and pathologic function, and where they converge on the same cellular pathways. Autopsy series point to distinct molecular actions as TDP-43 and FUS neuronal inclusions do not overlap, with FUS inclusions being present in only a small subgroup of patients. By contrast, modeling experiments in lower organisms support a genetic interaction between TDP-43 and FUS, although it is likely indirect. Regardless, the recent finding that additional RNA-binding proteins may also cause ALS, and the observation that TDP-43 aggregation remains a core feature in all of the recently identified genetic forms of ALS (C9ORF72, VCP, UBQLN2, and PFN1), underscores the central role of TDP-43 and RNA metabolism in ALS and FTLD.
Recent discoveries point to an unprecedented convergence of molecular pathways in ALS and FTLD involving RNA metabolism. Defining the exact points of convergence will likely be key to advancing therapeutics development in the coming years.
Intermediate filaments serve important structural roles, but other cellular functions are increasingly recognized. This study demonstrated normal function of the low‐molecular‐weight neurofilament ...protein (NFL) in mitochondrial dynamics and disruption in Charcot‐Marie‐Tooth disease (CMT) due to mutations in the Nefl gene. In motor neurons of spinal cord cultured from Nefl‐knockout mice, mitochondrial length and the rate of fusion were decreased concomitant with increased motility. These parameters were normalized after expression of NFLwt on the Nefl‐/‐background, but not by overexpression of the profusion protein, mitofusin 2 (MFN2). The effects of CMT‐causing NFL mutants bore similarities to and differences from Nefl knockout. In the early phase of toxicity before disruption of the neurofilament network, NFLQ333P and NFLP8R integrated into neurofilaments and had effects on mitochondria similar to those with Nefl knockout. The reduction of fusion rate by NFLQ333P was partly due to interference with the function of the profusion protein MFN2, which is mutated in CMT2A, functionally linking these forms of CMT. In the later phase of toxicity, mitochondria essentially stopped moving in neurons expressing NFL mutants, probably a consequence of cytoskeletal disruption. Overall, the data point to important functions of neurofilaments in mitochondrial dynamics as well as primary involvement in CMT2E/1F.—Gentil, B. J. Minotti, S., Beange, M., Baloh, R. H., Julien, J.‐P., Durham, H. D. Normal role of the low‐molecular‐weight neurofilament protein in mitochondrial dynamics and disruption in Charcot‐Marie‐Tooth disease. FASEB J. 26, 1194‐1203 (2012). www.fasebj.org
The GDNF family ligands (GFLs: GDNF, neurturin, persephin, and artemin) signal through RET and a glycosyl-phosphatidylinositol (GPI)-anchored coreceptor (GFRα1–α4) that binds ligand with high ...affinity and provides specificity. The importance of the GPI anchor is not fully understood; however, GPI-linked proteins cluster into lipid rafts, structures that may represent highly specialized signaling organelles. Here, we report that GPI-anchored GFRα1 recruits RET to lipid rafts after GDNF stimulation and results in RET/Src association. Disruption of RET localization using either transmembrane-anchored or soluble GFRα1 results in RET phosphorylation, but GDNF-induced intracellular signaling events are markedly attenuated as are neuronal differentiation and survival responses. Therefore, proper membrane localization of RET via interaction with a raft-localized, GPI-linked coreceptor is of fundamental importance in GFL signaling.
The glial cell line-derived neurotrophic factor (GDNF) ligands (GDNF, Neurturin NTN, and Persephin PSP) signal through a multicomponent receptor system composed of a high-affinity binding component ...(GFR alpha 1-GFR alpha 4) and a common signaling component (RET). Here, we report the identification of Artemin, a novel member of the GDNF family, and demonstrate that it is the ligand for the former orphan receptor GFR alpha 3-RET. Artemin is a survival factor for sensory and sympathetic neurons in culture, and its expression pattern suggests that it also influences these neurons in vivo. Artemin can also activate the GFR alpha 1-RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFR alpha 1-RET) and NTN (GFR alpha 2-RET), Artemin has a preferred receptor (GFR alpha 3-RET) but that alternative receptor interactions also occur.
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