Clonally expanded CD8 T cells characterize amyotrophic lateral sclerosis-4.

Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Revisiting Glutamate Excitotoxicity in Amyotrophic Lateral Sclerosis and Age-Related Neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest benefits. To design new and more effective therapies for ALS, particularly for sporadic ALS of unknown and diverse etiologies, we must identify key, convergent mechanisms of disease pathogenesis. This review focuses on the origin and effects of glutamate-mediated excitotoxicity in ALS (the cortical hyperexcitability hypothesis), in which increased glutamatergic signaling causes motor neurons to become hyperexcitable and eventually die. We characterize both primary and secondary contributions to excitotoxicity, referring to processes taking place at the synapse and within the cell, respectively. 'Primary pathways' include upregulation of calcium-permeable AMPA receptors, dysfunction of the EAAT2 astrocytic glutamate transporter, increased release of glutamate from the presynaptic terminal, and reduced inhibition by cortical interneurons-all of which have been observed in ALS patients and model systems. 'Secondary pathways' include changes to mitochondrial morphology and function, increased production of reactive oxygen species, and endoplasmic reticulum (ER) stress. By identifying key targets in the excitotoxicity cascade, we emphasize the importance of this pathway in the pathogenesis of ALS and suggest that intervening in this pathway could be effective for developing therapies for this disease.

Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients.

Amyotrophic lateral sclerosis type 4 (ALS4) is a rare, early-onset, autosomal dominant form of ALS, characterized by slow disease progression and sparing of respiratory musculature. Dominant, gain-of-function mutations in the senataxin gene (SETX) cause ALS4, but the mechanistic basis for motor neuron toxicity is unknown. SETX is a RNA-binding protein with a highly conserved helicase domain, but does not possess a low-complexity domain, making it unique among ALS-linked disease proteins. We derived ALS4 mouse models by expressing two different senataxin gene mutations (R2136H and L389S) via transgenesis and knock-in gene targeting. Both approaches yielded SETX mutant mice that develop neuromuscular phenotypes and motor neuron degeneration. Neuropathological characterization of SETX mice revealed nuclear clearing of TDP-43, accompanied by TDP-43 cytosolic mislocalization, consistent with the hallmark pathology observed in human ALS patients. Postmortem material from ALS4 patients exhibited TDP-43 mislocalization in spinal cord motor neurons, and motor neurons from SETX ALS4 mice displayed enhanced stress granule formation. Immunostaining analysis for nucleocytoplasmic transport proteins Ran and RanGAP1 uncovered nuclear membrane abnormalities in the motor neurons of SETX ALS4 mice, and nuclear import was delayed in SETX ALS4 cortical neurons, indicative of impaired nucleocytoplasmic trafficking. SETX ALS4 mice thus recapitulated ALS disease phenotypes in association with TDP-43 mislocalization and provided insight into the basis for TDP-43 histopathology, linking SETX dysfunction to common pathways of ALS motor neuron degeneration.

Senataxin helicase, the causal gene defect in ALS4, is a significant modifier of C9orf72 ALS G4C2 and arginine-containing dipeptide repeat toxicity.

Identifying genetic modifiers of familial amyotrophic lateral sclerosis (ALS) may reveal targets for therapeutic modulation with potential application to sporadic ALS. GGGGCC (G4C2) repeat expansions in the C9orf72 gene underlie the most common form of familial ALS, and generate toxic arginine-containing dipeptide repeats (DPRs), which interfere with membraneless organelles, such as the nucleolus. Here we considered senataxin (SETX), the genetic cause of ALS4, as a modifier of C9orf72 ALS, because SETX is a nuclear helicase that may regulate RNA-protein interactions involved in ALS dysfunction. After documenting that decreased SETX expression enhances arginine-containing DPR toxicity and C9orf72 repeat expansion toxicity in HEK293 cells and primary neurons, we generated SETX fly lines and evaluated the effect of SETX in flies expressing either (G4C2)58 repeats or glycine-arginine-50 [GR(50)] DPRs. We observed dramatic suppression of disease phenotypes in (G4C2)58 and GR(50) Drosophila models, and detected a striking relocalization of GR(50) out of the nucleolus in flies co-expressing SETX. Next-generation GR(1000) fly models, that show age-related motor deficits in climbing and movement assays, were similarly rescued with SETX co-expression. We noted that the physical interaction between SETX and arginine-containing DPRs is partially RNA-dependent. Finally, we directly assessed the nucleolus in cells expressing GR-DPRs, confirmed reduced mobility of proteins trafficking to the nucleolus upon GR-DPR expression, and found that SETX dosage modulated nucleolus liquidity in GR-DPR-expressing cells and motor neurons. These findings reveal a hitherto unknown connection between SETX function and cellular processes contributing to neuron demise in the most common form of familial ALS.

SUMOylated Senataxin functions in genome stability, RNA degradation, and stress granule disassembly, and is linked with inherited ataxia and motor neuron disease.

BACKGROUND: Senataxin (SETX) is a DNA/RNA helicase critical for neuron survival. SETX mutations underlie two inherited neurodegenerative diseases: Ataxia with Oculomotor Apraxia type 2 (AOA2) and Amyotrophic Lateral Sclerosis type 4 (ALS4). METHODS: This review examines SETX key cellular processes and we hypothesize that SETX requires SUMO posttranslational modification to function properly. RESULTS: SETX is localized to distinct foci during S-phase of the cell cycle, and these foci represent sites of DNA polymerase/RNA polymerase II (RNAP) collision, as they co-localize with DNA damage markers 53BP1 and H2AX. At such sites, SETX directs incomplete RNA transcripts to the nuclear exosome for degradation via interaction with exosome component 9 (Exosc9), a key component of the nuclear exosome. These processes require SETX SUMOylation. SETX was also recently localized within stress granules (SGs), and found to regulate SG disassembly, a process that similarly requires SUMOylation. CONCLUSION: SETX undergoes SUMO modification to function at S-phase foci in cycling cells to facilitate RNA degradation. SETX may regulate similar processes in non-dividing neurons at sites of RNAP II bidirectional self-collision. Finally, SUMOylation of SETX appears to be required for SG disassembly. This SETX function may be crucial for neuron survival, as altered SG dynamics are linked to ALS disease pathogenesis. In addition, AOA2 point mutations have been shown to block SETX SUMOylation. Such mutations induce an ataxia phenotype indistinguishable from those with SETX null mutation, underscoring the importance of this modification.

De novo pathogenic variant in SETX causes a rapidly progressive neurodegenerative disorder of early childhood-onset with severe axonal polyneuropathy.

Pathogenic variants in SETX cause two distinct neurological diseases, a loss-of-function recessive disorder, ataxia with oculomotor apraxia type 2 (AOA2), and a dominant gain-of-function motor neuron disorder, amyotrophic lateral sclerosis type 4 (ALS4). We identified two unrelated patients with the same de novo c.23C > T (p.Thr8Met) variant in SETX presenting with an early-onset, severe polyneuropathy. As rare private gene variation is often difficult to link to genetic neurological disease by DNA sequence alone, we used transcriptional network analysis to functionally validate these patients with severe de novo SETX-related neurodegenerative disorder. Weighted gene co-expression network analysis (WGCNA) was used to identify disease-associated modules from two different ALS4 mouse models and compared to confirmed ALS4 patient data to derive an ALS4-specific transcriptional signature. WGCNA of whole blood RNA-sequencing data from a patient with the p.Thr8Met SETX variant was compared to ALS4 and control patients to determine if this signature could be used to identify affected patients. WGCNA identified overlapping disease-associated modules in ALS4 mouse model data and ALS4 patient data. Mouse ALS4 disease-associated modules were not associated with AOA2 disease modules, confirming distinct disease-specific signatures. The expression profile of a patient carrying the c.23C > T (p.Thr8Met) variant was significantly associated with the human and mouse ALS4 signature, confirming the relationship between this SETX variant and disease. The similar clinical presentations of the two unrelated patients with the same de novo p.Thr8Met variant and the functional data provide strong evidence that the p.Thr8Met variant is pathogenic. The distinct phenotype expands the clinical spectrum of SETX-related disorders.

Tight expression regulation of senataxin, linked to motor neuron disease and ataxia, is required to avert cell-cycle block and nucleolus disassembly.

The Senataxin (SETX) protein exhibits strong sequence conservation with the helicase domain of the yeast protein Sen1p, and recessive SETX mutations cause a severe ataxia, known as Ataxia with Oculomotor Apraxia type 2, while dominant SETX mutations cause Amyotrophic Lateral Sclerosis type 4. SETX is a very low abundance protein, and its expression is tightly regulated, such that large increases in mRNA levels fail to significantly increase protein levels. Despite this, transient transfection in cell culture can boost SETX protein levels on an individual cell basis. Here we found that over-expression of normal SETX, but not enzymatically-dead SETX, is associated with S-phase cell-cycle arrest in HEK293A cells. As SETX interacts with the nuclear exosome to ensure degradation of incomplete RNA transcripts, and SETX localizes to sites of collision between the DNA replication machinery and the RNAP II complex, altered dosage or aberrant function of SETX may impede this process to promote S-phase cell-cycle arrest. Because neurons are enriched for long transcripts with additional antisense regulatory transcription, collisions of RNAP II complexes may occur in such post-mitotic cells, underscoring a role for SETX in maintaining neuron homeostasis.

Prevalence of ALDH7A1 mutations in 18 North American pyridoxine-dependent seizure (PDS) patients.

PURPOSE: Pyridoxine-dependent seizure (PDS) is a rare disorder characterized by seizures that are resistant to common anticonvulsants, and that are ultimately controlled by daily pharmacologic doses of pyridoxine (vitamin B6). Mutations of the antiquitin gene (ALDH7A1) are now recognized as the molecular basis of cases of neonatal-onset PDS. METHODS: Bidirectional DNA sequence analysis of ALDH7A1 was undertaken along with plasma pipecolic acid (PA) measurements to determine the prevalence of ALDH7A1 mutations in a cohort of 18 North American patients with PDS. RESULTS: In patients with neonatal-onset PDS, compound heterozygous or homozygous ALDH7A1 mutations were detected in 10 of 12 cases, and a single mutation was found in the remaining 2. In later-onset cases, mutations in ALDH7A1 were detected in three of six cases. In two patients with infantile spasms responsive to pyridoxine treatment and with good clinical outcomes, no mutations were found and PA levels were normal. In total, 13 novel mutations were identified. DISCUSSION: Our study advances previous findings that defects of ALDH7A1 are almost always the cause of neonatal-onset PDS and that defects in this gene are also responsible for some but not all later-onset cases. Later-onset cases of infantile spasms with good outcomes lacked evidence for antiquitin dysfunction, suggesting that this phenotype is less compelling for PDS.

Senataxin, A Novel Helicase at the Interface of RNA Transcriptome Regulation and Neurobiology: From Normal Function to Pathological Roles in Motor Neuron Disease and Cerebellar Degeneration.

Senataxin (SETX) is a DNA-RNA helicase whose C-terminal region shows homology to the helicase domain of the yeast protein Sen1p. Genetic discoveries have established the importance of SETX for neural function, as recessive mutations in the SETX gene cause Ataxia with Oculomotor Apraxia type 2 (AOA2) (OMIM: 606002), which is the third most common form of recessive ataxia, after Friedreich's ataxia and Ataxia-Telangiectasia. In addition, rare, dominant SETX mutations cause a juvenile-onset form of Amyotrophic Lateral Sclerosis (ALS), known as ALS4. SETX performs a number of RNA regulatory functions, including maintaining RNA transcriptome homeostasis. Over the last decade, altered RNA regulation and aberrant RNA-binding protein function have emerged as a central theme in motor neuron disease pathogenesis, with evidence suggesting that sporadic ALS disease pathology may overlap with the molecular pathology uncovered in familial ALS. Like other RNA processing proteins linked to ALS, the basis for SETX gain-of-function motor neuron toxicity remains ill-defined. Studies of yeast Sen1p and mammalian SETX protein have revealed a range of important RNA regulatory functions, including resolution of R-loops to permit transcription termination, and RNA splicing. Growing evidence suggests that SETX may represent an important genetic modifier locus for sporadic ALS. In cycling cells, SETX is found at nuclear foci during the S/G2 cell-cycle transition phase, and may function at sites of collision between components of the replisome and transcription machinery. While we do not yet know which SETX activities are most critical to neurodegeneration, our evolving understanding of SETX function will undoubtedly be crucial for not only understanding the role of SETX in ALS and ataxia disease pathogenesis, but also for delineating the mechanistic biology of fundamentally important molecular processes in the cell.

Unwinding the role of senataxin in neurodegeneration.

Interest in senataxin biology began in 2004 when mutations were first identified in what was then a novel protein. Dominantly inherited mutations were documented in rare juvenile-onset, motor neuron disease pedigrees in a familial form of amyotrophic lateral sclerosis (ALS4), while recessive mutations were found to cause a severe early-onset ataxia with oculomotor apraxia (AOA2) that is actually the second most common recessive ataxia after Freidreich's ataxia. From earlier studies of sen1p, the yeast ortholog of senataxin, a range of important RNA processing functions have been attributed to this protein. Like sen1p, senataxin contains a helicase domain to interact with RNA and an amino-terminal domain for critical protein interactions. Senataxin also joins a group of important proteins responsible for maintaining RNA transcriptome homeostasis, including FUS, TDP-43, and SMN that can all cause familial forms of motor neuron disease (MND). Independent of this association, senataxin is gaining attention for its role in maintaining genomic stability. Senataxin has been shown to resolve R-Loop structures, which form when nascent RNA hybridizes to DNA, displacing the non-transcribed strand. But in cycling cells, senataxin is also found at nuclear foci during the S/G2 cell-cycle phase, and may function at sites of specific collision between components of the replisome and transcription machinery. Which of these important processes is most critical to prevent neurodegeneration remains unknown, but our evolving understanding of these processes will be crucial not only for understanding senataxin's role in neurological disease, but also in a number of fundamentally important cellular functions.

Search for genes involved in Joubert syndrome: evidence that one or more major loci are yet to be identified and exclusion of candidate genes EN1, EN2, FGF8, and BARHL1.

Joubert syndrome (JS) is a rare autosomal recessive malformation syndrome involving agenesis or dysgenesis of the cerebellar vermis with accompanying brainstem malformations. JS is further characterized by hypotonia, developmental delay, intermittent hyperpnea, and abnormal eye movements. The biochemical and molecular basis of JS remains unknown, although several genes that are crucial in the development of the cerebellum have been proposed as attractive candidate genes. JS is clinically heterogeneous; this, together with previous linkage analyses, suggests that there may also be genetic heterogeneity. A locus for JS was previously identified on chromosome 9q34 by linkage analysis in a consanguineous family of Arabian origin. A putative second JS locus was recently suggested when a deletion on chromosome 17p11.2 was observed in a patient with Smith-Magenis syndrome and JS phenotype. We have investigated a cohort of apparently unrelated North American JS pedigrees for association with the loci on chromosomes 9q34 and 17p11.2 and excluded them in all cases where data were informative. Analysis of an additional 21 unrelated JS patients showed no evidence of homozygosity at the 9q34 and 17p11.2 loci that would suggest inheritance of founder JS mutation(s) or unreported consanguinity. Together, these data suggest that one or more major loci for JS remain to be identified. Consequently, we undertook mutation analysis of several functional candidate genes, EN1, EN2, and FGF8, in a total of 26 unrelated JS patients. Our data suggest that all of these genes may be excluded from a direct pathogenic role in JS. The BARHL1 gene, which localizes to chromosome 9q34 and has previously been proposed as a strong positional candidate gene for JS, was also investigated and excluded from involvement in JS that is linked to chromosome 9q34.

Microtubule defects & Neurodegeneration.

One of the major challenges facing the long term survival of neurons is their requirement to maintain efficient axonal transport over long distances. In humans as large, long-lived vertebrates, the machinery maintaining neuronal transport must remain efficient despite the slow accumulation of cell damage during aging. Mutations in genes encoding proteins which function in the transport system feature prominently in neurologic disorders. Genes known to cause such disorders and showing traditional Mendelian inheritance have been more readily identified. It has been more difficult, however, to isolate factors underlying the complex genetics contributing to the more common idiopathic forms of neurodegenerative disease. At the heart of neuronal transport is the rail network or scaffolding provided by neuron specific microtubules (MTs). The importance of MT dynamics and stability is underscored by the critical role tau protein plays in MT-associated stabilization versus the dysfunction seen in Alzheimer's disease, frontotemporal dementia and other tauopathies. Another example of the requirement for tight regulation of MT dynamics is the need to maintain balanced levels of post-translational modification of key MT building-blocks such as α-tubulin. Tubulins require extensive polyglutamylation at their carboxyl-terminus as part of a novel post-translational modification mechanism to signal MT growth versus destabilization. Dramatically, knock-out of a gene encoding a deglutamylation family member causes an extremely rapid cell death of Purkinje cells in the ataxic mouse model, pcd. This review will examine a range of neurodegenerative conditions where current molecular understanding points to defects in the stability of MTs and axonal transport to emphasize the central role of MTs in neuron survival.

Protein interaction analysis of senataxin and the ALS4 L389S mutant yields insights into senataxin post-translational modification and uncovers mutant-specific binding with a brain cytoplasmic RNA-encoded peptide.

Senataxin is a large 303 kDa protein linked to neuron survival, as recessive mutations cause Ataxia with Oculomotor Apraxia type 2 (AOA2), and dominant mutations cause amyotrophic lateral sclerosis type 4 (ALS4). Senataxin contains an amino-terminal protein-interaction domain and a carboxy-terminal DNA/RNA helicase domain. In this study, we focused upon the common ALS4 mutation, L389S, by performing yeast two-hybrid screens of a human brain expression library with control senataxin or L389S senataxin as bait. Interacting clones identified from the two screens were collated, and redundant hits and false positives subtracted to yield a set of 13 protein interactors. Among these hits, we discovered a highly specific and reproducible interaction of L389S senataxin with a peptide encoded by the antisense sequence of a brain-specific non-coding RNA, known as BCYRN1. We further found that L389S senataxin interacts with other proteins containing regions of conserved homology with the BCYRN1 reverse complement-encoded peptide, suggesting that such aberrant protein interactions may contribute to L389S ALS4 disease pathogenesis. As the yeast two-hybrid screen also demonstrated senataxin self-association, we confirmed senataxin dimerization via its amino-terminal binding domain and determined that the L389S mutation does not abrogate senataxin self-association. Finally, based upon detection of interactions between senataxin and ubiquitin-SUMO pathway modification enzymes, we examined senataxin for the presence of ubiquitin and SUMO monomers, and observed this post-translational modification. Our senataxin protein interaction study reveals a number of features of senataxin biology that shed light on senataxin normal function and likely on senataxin molecular pathology in ALS4.

Systematic review of TCF2 anomalies in renal cysts and diabetes syndrome/maturity onset diabetes of the young type 5.

OBJECTIVE: There is a paucity of published works that systematically evaluate gene anomalies or clinical features of patients with renal cysts and diabetes syndrome (RCAD)/maturity onset diabetes of the young type 5 (MODY5). The purpose of this review was to systematically assess the detection rate, genetic and phenotypic implications of heterozygous autosomal dominant TCF2 anomalies. DATA SOURCES: MEDLINE database was searched to select articles recorded in English from 1997 to 2008. The focus was monoallelic germline TCF2 gene mutations/deletions. Biallelic inactivation, polymorphisms, DNA modification (hypomethylation and hypermethylation), loci associated with cancer risk, and somatic TCF2 anomalies were all excluded. STUDY SELECTION: After searching the literature, 50 articles were selected. RESULTS: The detection rate of TCF2 anomalies was 9.7% and varied considerably among MODY (1.4%), renal structure anomalies (RSA) (21.4%) and RSA with MODY (41.2%) subgroups. Mutations were strikingly located within the DNA binding domain and varied among exons of the DNA binding domain: exons 2 and 4 were the hottest spots, while mutations were sporadically distributed in exon 3. The consistent phenotypes were RSA (89.6%) and diabetes mellitus (DM) (45.0%). However, the concurrence of RSA and DM was relatively low (27.5%), which hinders the optimal performance of genetic testing and obtainment of timely diagnosis. Other organ involvements were complementary and necessary for the early identification of patients with TCF2 anomalies. Analysis of phenotypes of TCF2 point mutations showed significant differences in the detection rates of RSA, impaired renal function (IRF) and DM according to mutation type but not mutation location. CONCLUSION: These valuable features of TCF2 anomalies that previously did not receive sufficient attention should not be neglected.

Mitochondrial dysfunction in NnaD mutant flies and Purkinje cell degeneration mice reveals a role for Nna proteins in neuronal bioenergetics.

The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in 3 weeks. Loss of function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 ortholog (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD/Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss of function results in altered bioenergetics and mitochondrial dysfunction.

Senataxin, the yeast Sen1p orthologue: characterization of a unique protein in which recessive mutations cause ataxia and dominant mutations cause motor neuron disease.

A severe recessive cerebellar ataxia, Ataxia-Oculomotor Apraxia 2 (AOA2) and a juvenile onset form of dominant amyotrophic lateral sclerosis (ALS4) result from mutations of the Senataxin (SETX) gene. To begin characterization this disease protein, we developed a specific antibody to the DNA/RNA helicase domain of SETX. In murine brain, SETX concentrates in several regions, including cerebellum, hippocampus and olfactory bulb with a general neuronal expression profile, colocalizing with NeuN. In cultured cells, we found that SETX was cytoplasmically diffuse, but in the nucleus, SETX was punctate, colocalizing with fibrillarin, a marker of the nucleolus. In differentiated non-cycling cells, nuclear SETX was not restricted to the nucleolus but was diffuse within the nucleoplasm, suggesting cell-cycle-dependent localization. SETX missense mutations cluster within the N-terminus and helicase domains. Flag tagging at the N-terminus caused protein mislocation to the nucleoplasm and failure to export to the cytoplasm, suggesting that the N-terminus may be essential for correct SETX localization. We report here the first characterization of SETX protein, which may provide future insights into a new mechanism leading to neuron death.

SIMPLE interacts with NEDD4 and TSG101: evidence for a role in lysosomal sorting and implications for Charcot-Marie-Tooth disease.

Mutation of the SIMPLE gene (small integral membrane protein of the lysosome/late endosome) is the molecular basis of Charcot-Marie-Tooth disease type 1C (CMT1C), a demyelinating peripheral neuropathy. Although the precise function of SIMPLE is unknown, prior reports suggest it localizes to the lysosome/late endosome. Furthermore, murine Simple interacts with Nedd4 (neural precursor cell expressed, developmentally downregulated 4), an E3 ubiquitin ligase that is important for regulating lysosomal degradation of plasma membrane proteins. To bring insights into the biochemical function of human SIMPLE, we confirmed that human SIMPLE interacts with NEDD4 and also report a novel interaction with tumor susceptibility gene 101 (TSG101), a class E vacuolar sorting protein. TSG101 is known to function downstream of NEDD4, sorting ubiquitinated substrates into multivesicular bodies (MVBs), which then deliver their cargo into the lysosomal lumen for degradation. Given the interaction with NEDD4 and TSG101, and the localization of SIMPLE along the lysosomal degradation pathway, we hypothesize that SIMPLE plays a role in the lysosomal sorting of plasma membrane proteins. We examine three CMT1C-associated SIMPLE mutations and show that they do not affect the interaction with NEDD4 or TSG101, nor do they lead to altered subcellular localization.

Joubert syndrome: a haplotype segregation strategy and exclusion of the zinc finger protein of cerebellum 1 (ZIC1) gene.

Joubert syndrome (JS) is a rare autosomal recessive malformation syndrome, involving dysgenesis of the cerebellar vermis with accompanying brainstem malformations (comprising the molar tooth sign). JS is characterized by hypotonia, developmental delay, intermittent hyperpnea and apnea, and abnormal eye movements. A single locus for JS was previously identified on 9q34 in a consanguineous family of Arabian origin. However, linkage to this locus has subsequently been shown to be rare. We have ascertained 35 JS pedigrees for haplotype segregation analysis of genetic loci for genes with a putative role in cerebellar development. We examined the ZIC1 gene as a functional candidate for JS as Zic1(-/-) null mice have a phenotype reminiscent of JS. We undertook mutational analysis of ZIC1 by standard mutational analysis (dideoxy-fingerprinting (ddf)) of 47 JS probands, and fully sequenced the coding region in five of these probands. By these means, ZIC1 was excluded from playing a causal role in most cases of JS as no disease-associated mutations were identified. Further, linkage to the ZIC1 genetic locus (3q24) was excluded in 21 of 35 pedigrees by haplotype segregation analysis of closely spaced markers. The remaining 14 of 35 pedigrees were consistent with linkage. However, this number does not significantly depart from that expected by random chance (16.5) for this cohort. Therefore, this systematic approach has been validated as a means to prioritize functional candidate genes and enables us to confine mutational analysis to only those probands whose segregation is consistent with linkage to any given locus.