Robert H. Baloh

Artemin, a Novel Member of the GDNF Ligand Family, Supports Peripheral and Central Neurons and Signals through the GFRα3–RET Receptor Complex

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 (GFRalpha1-GFRalpha4) 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 GFRalpha3-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 GFRalpha1-RET complex and supports the survival of dopaminergic midbrain neurons in culture, indicating that like GDNF (GFRalpha1-RET) and NTN (GFRalpha2-RET), Artemin has a preferred receptor (GFRalpha3-RET) but that alternative receptor interactions also occur.

Persephin, a Novel Neurotrophic Factor Related to GDNF and Neurturin

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

TDP-43 A315T mutation in familial motor neuron disease

To identify novel causes of familial neurodegenerative diseases, we extended our previous studies of TAR DNA‐binding protein 43 (TDP‐43) proteinopathies to investigate TDP‐43 as a candidate gene in familial cases of motor neuron disease. Sequencing of the TDP‐43 gene led to the identification of a novel missense mutation, Ala‐315‐Thr, which segregates with all affected members of an autosomal dominant motor neuron disease family. The mutation was not found in 1,505 healthy control subjects. The discovery of a missense mutation in TDP‐43 in a family with dominantly inherited motor neuron disease provides evidence of a direct link between altered TDP‐43 function and neurodegeneration. Ann Neurol 2008

TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases that show considerable clinical and pathologic overlap, with no effective treatments available. Mutations in the RNA binding protein TDP-43 were recently identified in patients with familial amyotrophic lateral sclerosis (ALS), and TDP-43 aggregates are found in both ALS and FTLD-U (FTLD with ubiquitin aggregates), suggesting a common underlying mechanism. We report that mice expressing a mutant form of human TDP-43 develop a progressive and fatal neurodegenerative disease reminiscent of both ALS and FTLD-U. Despite universal transgene expression throughout the nervous system, pathologic aggregates of ubiquitinated proteins accumulate only in specific neuronal populations, including layer 5 pyramidal neurons in frontal cortex, as well as spinal motor neurons, recapitulating the phenomenon of selective vulnerability seen in patients with FTLD-U and ALS. Surprisingly, cytoplasmic TDP-43 aggregates are not present, and hence are not required for TDP-43-induced neurodegeneration. These results indicate that the cellular and molecular substrates for selective vulnerability in FTLD-U and ALS are shared between mice and humans, and suggest that altered DNA/RNA-binding protein function, rather than toxic aggregation, is central to TDP-43-related neurodegeneration.

The GDNF family ligands and receptors — implications for neural development

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.

Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways

New players in Lou Gehrigs disease Amyotrophic lateral sclerosis (ALS), often referred to as “Lou Gehrigs disease,” is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. Cirulli et al. sequenced the expressed genes of nearly 3000 ALS patients and compared them with those of more than 6000 controls (see the Perspective by Singleton and Traynor). They identified several proteins that were linked to disease in patients. One such protein, TBK1, is implicated in innate immunity and autophagy and may represent a therapeutic target. Science, this issue p. 1436; see also p. 1422 Analysis of the expressed genes of nearly 2900 patients with amyotrophic lateral sclerosis and about 6400 controls reveals a disease predisposition–associated gene. [Also see Perspective by Singleton and Traynor] Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease with no effective treatment. We report the results of a moderate-scale sequencing study aimed at increasing the number of genes known to contribute to predisposition for ALS. We performed whole-exome sequencing of 2869 ALS patients and 6405 controls. Several known ALS genes were found to be associated, and TBK1 (the gene encoding TANK-binding kinase 1) was identified as an ALS gene. TBK1 is known to bind to and phosphorylate a number of proteins involved in innate immunity and autophagy, including optineurin (OPTN) and p62 (SQSTM1/sequestosome), both of which have also been implicated in ALS. These observations reveal a key role of the autophagic pathway in ALS and suggest specific targets for therapeutic intervention.

TrnR2, a novel receptor that mediates neurturin and GDNF signaling through Ret

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) comprise a family of TGF-beta-related neurotrophic factors (TRNs), which have trophic influences on a variety of neuronal populations. A receptor complex comprised of TrnR1 (GDNFR alpha) and Ret was recently identified and found to be capable of mediating both GDNF and NTN signaling. We have identified a novel receptor based on homology to TrnR1, called TrnR2, that is 48% identical to TrnR1, and is located on the short arm of chromosome 8. TrnR2 is attached to the cell surface via a GPI-linkage, and can mediate both NTN and GDNF signaling through Ret in vitro. Fibroblasts expressing TrnR2 and Ret are approximately 30-fold more sensitive to NTN than to GDNF treatment, whereas those expressing TrnR1 and Ret respond equivalently to both factors, suggesting the TrnR2-Ret complex acts preferentially as a receptor for NTN. TrnR2 and Ret are expressed in neurons of the superior cervical and dorsal root ganglia, and in the adult brain. Comparative analysis of TrnR1, TrnR2, and Ret expression indicates that multiple receptor complexes, capable of mediating GDNF and NTN signaling, exist in vivo.

Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex

Mitofusins (Mfn1 and Mfn2) are outer mitochondrial membrane proteins involved in regulating mitochondrial dynamics. Mutations in Mfn2 cause Charcot-Marie-Tooth disease (CMT) type 2A, an inherited disease characterized by degeneration of long peripheral axons, but the nature of this tissue selectivity remains unknown. Here, we present evidence that Mfn2 is directly involved in and required for axonal mitochondrial transport, distinct from its role in mitochondrial fusion. Live imaging of neurons cultured from Mfn2 knock-out mice or neurons expressing Mfn2 disease mutants shows that axonal mitochondria spend more time paused and undergo slower anterograde and retrograde movements, indicating an alteration in attachment to microtubule-based transport systems. Furthermore, Mfn2 disruption altered mitochondrial movement selectively, leaving transport of other organelles intact. Importantly, both Mfn1 and Mfn2 interact with mammalian Miro (Miro1/Miro2) and Milton (OIP106/GRIF1) proteins, members of the molecular complex that links mitochondria to kinesin motors. Knockdown of Miro2 in cultured neurons produced transport deficits identical to loss of Mfn2, indicating that both proteins must be present at the outer membrane to mediate axonal mitochondrial transport. In contrast, disruption of mitochondrial fusion via knockdown of the inner mitochondrial membrane protein Opa1 had no effect on mitochondrial motility, indicating that loss of fusion does not inherently alter mitochondrial transport. These experiments identify a role for mitofusins in directly regulating mitochondrial transport and offer important insight into the cell type specificity and molecular mechanisms of axonal degeneration in CMT2A and dominant optic atrophy.

Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion

Antisense oligonucleotides can correct disease-specific phenotypes in cultured motor neurons differentiated from iPSCs derived from ALS patients with a C9ORF72 repeat expansion. Clearing Toxic RNA in ALS Amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease) is a uniformly fatal disease caused by the death of cells in the nervous system that control the musculature. Patients slowly become paralyzed and lose the ability to breathe, and no effective therapies currently exist. The expansion of a repeated DNA element (GGGGCC) in a gene called C9ORF72 was recently identified as the most common genetic cause of ALS. In their new study, Sareen et al. set out to understand how the expansion of the GGGGCC repeat in C9ORF72 causes cell degeneration. They took skin cells from patients with the disease and converted them into motor neurons in a culture dish, the cells that die in ALS patients. They found that large pieces of RNA containing the expanded GGGGCC repeat built up in neurons from ALS patients and disrupted the function of these cells. Furthermore, they observed that oligonucleotides complementary to the C9ORF72 RNA transcript sequence (“antisense oligonucleotides”) suppressed the formation of these RNA foci. These findings support the idea that the buildup of “toxic” RNA containing the GGGGCC repeat contributes to the death of motor neurons in ALS, and suggest that antisense oligonucleotides targeting this transcript may be a strategy for treating ALS patients with the C9ORF72 repeat expansion. 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.

Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease

Accumulation of autophagosomes because of impaired autophagy during valosin-containing protein (VCP)–linked dementia is explained by the absence or reduced activity of VCP.