Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia.

Amyotrophic lateral sclerosis (ALS) is a paralytic and usually fatal disorder caused by motor-neuron degeneration in the brain and spinal cord. Most cases of ALS are sporadic but about 5-10% are familial. Mutations in superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP, also known as TDP43) and fused in sarcoma (FUS, also known as translocated in liposarcoma (TLS)) account for approximately 30% of classic familial ALS. Mutations in several other genes have also been reported as rare causes of ALS or ALS-like syndromes. The causes of the remaining cases of familial ALS and of the vast majority of sporadic ALS are unknown. Despite extensive studies of previously identified ALS-causing genes, the pathogenic mechanism underlying motor-neuron degeneration in ALS remains largely obscure. Dementia, usually of the frontotemporal lobar type, may occur in some ALS cases. It is unclear whether ALS and dementia share common aetiology and pathogenesis in ALS/dementia. Here we show that mutations in UBQLN2, which encodes the ubiquitin-like protein ubiquilin 2, cause dominantly inherited, chromosome-X-linked ALS and ALS/dementia. We describe novel ubiquilin 2 pathology in the spinal cords of ALS cases and in the brains of ALS/dementia cases with or without UBQLN2 mutations. Ubiquilin 2 is a member of the ubiquilin family, which regulates the degradation of ubiquitinated proteins. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation. Therefore, our findings link abnormalities in ubiquilin 2 to defects in the protein degradation pathway, abnormal protein aggregation and neurodegeneration, indicating a common pathogenic mechanism that can be exploited for therapeutic intervention.

Dendritic spinopathy in transgenic mice expressing ALS/dementia-linked mutant UBQLN2.

Mutations in the gene encoding ubiquilin2 (UBQLN2) cause amyotrophic lateral sclerosis (ALS), frontotemporal type of dementia, or both. However, the molecular mechanisms are unknown. Here, we show that ALS/dementia-linked UBQLN2(P497H) transgenic mice develop neuronal pathology with ubiquilin2/ubiquitin/p62-positive inclusions in the brain, especially in the hippocampus, recapitulating several key pathological features of dementia observed in human patients with UBQLN2 mutations. A major feature of the ubiquilin2-related pathology in these mice, and reminiscent of human disease, is a dendritic spinopathy with protein aggregation in the dendritic spines and an associated decrease in dendritic spine density and synaptic dysfunction. Finally, we show that the protein inclusions in the dendritic spines are composed of several components of the proteasome machinery, including Ub(G76V)-GFP, a representative ubiquitinated protein substrate that is accumulated in the transgenic mice. Our data, therefore, directly link impaired protein degradation to inclusion formation that is associated with synaptic dysfunction and cognitive deficits. These data imply a convergent molecular pathway involving synaptic protein recycling that may also be involved in other neurodegenerative disorders, with implications for development of widely applicable rational therapeutics.

Mutation in the novel nuclear-encoded mitochondrial protein CHCHD10 in a family with autosomal dominant mitochondrial myopathy.

Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutations in either the mitochondrial or nuclear genome. Only 5% of all mitochondrial disorders are autosomal dominant. We analyzed DNA from members of the previously reported Puerto Rican kindred with an autosomal dominant mitochondrial myopathy (Heimann-Patterson et al. 1997). Linkage analysis suggested a putative locus on the pericentric region of the long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we established chromosome 22 open reading frame 16 (C22orf16) (later designated as CHCHD10) as the only high-scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double-missense mutation (R15S and G58R) in cis in CHCHD10 which encodes a coiled coil-helix-coiled coil-helix protein of unknown function. These two mutations completely co-segregated with the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic (including 32 Puerto Rican) controls. Expression profiling showed that CHCHD10 is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10 protein was confirmed using immunofluorescence in cells expressing either wild-type or mutant CHCHD10. We found that the expression of the G58R, but not the R15S, mutation induced mitochondrial fragmentation. Our findings identify a novel gene causing mitochondrial myopathy, thereby expanding the spectrum of mitochondrial myopathies caused by nuclear genes. Our findings also suggest a role for CHCHD10 in the morphologic remodeling of the mitochondria.

Mutant TRPV4-mediated toxicity is linked to increased constitutive function in axonal neuropathies.

Mutations in TRPV4 have been linked to three distinct axonal neuropathies. However, the pathogenic mechanism underlying these disorders remains unclear. Both gain and loss of calcium channel activity of the mutant TRPV4 have been suggested. Here, we show that the three previously reported TRPV4 mutant channels have a physiological localization and display an increased calcium channel activity, leading to increased cytotoxicity in three different cell types. Patch clamp experiments showed that cells expressing mutant TRPV4 have much larger whole-cell currents than those expressing the wild-type TRPV4 channel. Single channel recordings showed that the mutant channels have higher open probability, due to a modification of gating, and no change in single-channel conductance. These data support the hypothesis that a "gain of function" mechanism, possibly leading to increased intracellular calcium influx, underlies the pathogenesis of the TRPV4-linked axonal neuropathies, and may have immediate implications for designing rational therapies.

UBQLN2/P62 cellular recycling pathways in amyotrophic lateral sclerosis and frontotemporal dementia.

Recent findings highlight a pathologic and functional convergence in amyotrophic lateral sclerosis (ALS) and amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD) at the level of protein recycling and disposal. Genes linked to rare cases of familial ALS and ALS-FTD, like UBQLN2, OPTN, SQSTM1/p62, and VCP, may converge onto a unifying pathogenic pathway and thereby provide novel therapeutic targets common to a spectrum of etiologically diverse forms of ALS and ALS-FTD. Interactions between these genes need to be further explored to understand their common molecular pathways. Future efforts should be directed toward generation and characterization of in vivo models to dissect the pathogenic mechanisms of ALS and ALS-FTD and the role of protein degradation pathways, both centrally, at the cell body, and peripherally, at the level of the synapse. Such efforts will rapidly accelerate the discovery of new drugs that regulate accumulation of pathogenic proteins and their downstream consequences in ALS and ALS-FTD and, possibly, other neurodegenerative diseases as well.

FUS-immunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal disorder of motor neuron degeneration. Most cases of ALS are sporadic (SALS), but about 5 to 10% of ALS cases are familial (FALS). Recent studies have shown that mutations in FUS are causal in approximately 4 to 5% of FALS and some apparent SALS cases. The pathogenic mechanism of the mutant FUS-mediated ALS and potential roles of FUS in non-FUS ALS remain to be investigated. METHODS: Immunostaining was performed on postmortem spinal cords from 78 ALS cases, including SALS (n = 52), ALS with dementia (ALS/dementia, n = 10), and FALS (n = 16). In addition, postmortem brains or spinal cords from 22 cases with or without frontotemporal lobar degeneration were also studied. In total, 100 cases were studied. RESULTS: FUS-immunoreactive inclusions were observed in spinal anterior horn neurons in all SALS and FALS cases, except for those with SOD1 mutations. The FUS-containing inclusions were also immunoreactive with antibodies to TDP43, p62, and ubiquitin. A fraction of tested FUS antibodies recognized FUS inclusions, and specific antigen retrieval protocol appeared to be important for detection of the skein-like FUS inclusions. INTERPRETATION: Although mutations in FUS account for only a small fraction of FALS and SALS, our data suggest that FUS protein may be a common component of the cellular inclusions in non-SOD1 ALS and some other neurodegenerative conditions, implying a shared pathogenic pathway underlying SALS, non-SOD1 FALS, ALS/dementia, and related disorders. Our data also indicate that SOD1-linked ALS may have a pathogenic pathway distinct from SALS and other types of FALS.

An unusual case of familial ALS and cerebellar ataxia.

We report a case of familial amyotrophic lateral sclerosis (FALS) with clinical signs of cerebellar and posterior column involvement. The patient's work-up showed a known mutation (E100K) in the gene for Cu/Zn superoxide dismutase 1 (SOD1). Our case illustrates that extramotor symptoms, such as prominent cerebellar signs, can be seen in patients with FALS.

Identification of TMEM230 mutations in familial Parkinson's disease.

Parkinson's disease is the second most common neurodegenerative disorder without effective treatment. It is generally sporadic with unknown etiology. However, genetic studies of rare familial forms have led to the identification of mutations in several genes, which are linked to typical Parkinson's disease or parkinsonian disorders. The pathogenesis of Parkinson's disease remains largely elusive. Here we report a locus for autosomal dominant, clinically typical and Lewy body-confirmed Parkinson's disease on the short arm of chromosome 20 (20pter-p12) and identify TMEM230 as the disease-causing gene. We show that TMEM230 encodes a transmembrane protein of secretory/recycling vesicles, including synaptic vesicles in neurons. Disease-linked TMEM230 mutants impair synaptic vesicle trafficking. Our data provide genetic evidence that a mutant transmembrane protein of synaptic vesicles in neurons is etiologically linked to Parkinson's disease, with implications for understanding the pathogenic mechanism of Parkinson's disease and for developing rational therapies.

Protein recycling pathways in neurodegenerative diseases.

Many progressive neurodegenerative diseases, including Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, and frontotemporal lobe dementia, are associated with the formation of insoluble intracellular proteinaceous inclusions. It is therefore imperative to understand the factors that regulate normal, as well as abnormal, protein recycling in neurons. Dysfunction of the ubiquitin-proteasome or autophagy pathways might contribute to the pathology of various neurodegenerative diseases. Induction of these pathways may offer a rational therapeutic strategy for a number of these diseases.

Making connections: pathology and genetics link amyotrophic lateral sclerosis with frontotemporal lobe dementia.

Over the last couple of decades, there has been a growing body of clinical, genetic, and histopathological evidence that similar pathological processes underlie amyotrophic lateral sclerosis (ALS) and some types of frontotemporal lobe dementia (FTD). Even though there is great diversity in the genetic causes of these disorders, there is a high degree of overlap in their histopathology. Genes linked to rare cases of familial ALS and/or FTD, like FUS, TARDBP, OPTN, and UBQLN2 may converge onto a unifying pathogenic pathway and thereby provide novel therapeutic targets common to a spectrum of etiologically diverse forms of ALS and ALS-FTD. Additionally, there are major loci for ALS-FTD on chromosomes 9p and 15q. Identification of causative genetic alterations at those loci will be an important step in understanding the pathogenesis of juvenile- and adult-onset ALS and ALS-FTD. Interactions between TDP-43, FUS, optineurin, and ubiquilin 2 need to be studied to understand their common molecular pathways. Future efforts should also be directed towards generation and characterization of in vivo models to dissect the pathogenic mechanisms of these diseases. Such efforts will rapidly accelerate the discovery of new drugs that regulate accumulation of pathogenic proteins and their downstream consequences.

Differential involvement of optineurin in amyotrophic lateral sclerosis with or without SOD1 mutations.

BACKGROUND: Mutations in optineurin have recently been linked to amyotrophic lateral sclerosis (ALS). OBJECTIVE: To determine whether optineurin-positive skeinlike inclusions are a common pathologic feature in ALS, including SOD1 -linked ALS. DESIGN: Clinical case series. SETTING: Academic referral center. SUBJECTS: We analyzed spinal cord sections from 46 clinically and pathologically diagnosed ALS cases and ALS transgenic mouse models overexpressing ALS-linked SOD1 mutations G93A or L126Z. RESULTS: We observed optineurin-immunoreactive skeinlike inclusions in all the sporadic ALS and familial ALS cases without SOD1 mutation, but not in cases with SOD1 mutations or in transgenic mice overexpressing the ALS-linked SOD1 mutations G93A or L126Z. CONCLUSION: The data from this study provide evidence that optineurin is involved in the pathogenesis of sporadic ALS and non- SOD1 familial ALS, thus supporting the hypothesis that these forms of ALS share a pathway that is distinct from that of SOD1-linked ALS.

SQSTM1 mutations in familial and sporadic amyotrophic lateral sclerosis.

BACKGROUND: The SQSTM1 gene encodes p62, a major pathologic protein involved in neurodegeneration. OBJECTIVE: To examine whether SQSTM1 mutations contribute to familial and sporadic amyotrophic lateral sclerosis (ALS). DESIGN: Case-control study. SETTING: Academic research. Patients  A cohort of 546 patients with familial (n = 340) or sporadic (n = 206) ALS seen at a major academic referral center were screened for SQSTM1 mutations. MAIN OUTCOME MEASURES: We evaluated the distribution of missense, deletion, silent, and intronic variants in SQSTM1 among our cohort of patients with ALS. In silico analysis of variants was performed to predict alterations in p62 structure and function. RESULTS: We identified 10 novel SQSTM1 mutations (9 heterozygous missense and 1 deletion) in 15 patients (6 with familial ALS and 9 with sporadic ALS). Predictive in silico analysis classified 8 of 9 missense variants as pathogenic. CONCLUSIONS: Using candidate gene identification based on prior biological knowledge and the functional prediction of rare variants, we identified several novel SQSTM1 mutations in patients with ALS. Our findings provide evidence of a direct genetic role for p62 in ALS pathogenesis and suggest that regulation of protein degradation pathways may represent an important therapeutic target in motor neuron degeneration.

Scapuloperoneal spinal muscular atrophy and CMT2C are allelic disorders caused by alterations in TRPV4.

Scapuloperoneal spinal muscular atrophy (SPSMA) and hereditary motor and sensory neuropathy type IIC (HMSN IIC, also known as HMSN2C or Charcot-Marie-Tooth disease type 2C (CMT2C)) are phenotypically heterogeneous disorders involving topographically distinct nerves and muscles. We originally described a large New England family of French-Canadian origin with SPSMA and an American family of English and Scottish descent with CMT2C. We mapped SPSMA and CMT2C risk loci to 12q24.1-q24.31 with an overlapping region between the two diseases. Further analysis reduced the CMT2C risk locus to a 4-Mb region. Here we report that SPSMA and CMT2C are allelic disorders caused by mutations in the gene encoding the transient receptor potential cation channel, subfamily V, member 4 (TRPV4). Functional analysis revealed that increased calcium channel activity is a distinct property of both SPSMA- and CMT2C-causing mutant proteins. Our findings link mutations in TRPV4 to altered calcium homeostasis and peripheral neuropathies, implying a pathogenic mechanism and possible options for therapy for these disorders.

Signaling mechanisms mediated by G-protein coupled receptors in human platelets.

AIM: The present study deals with the investigation of mechanisms involved in the synergistic interaction between epinephrine and arachidonic acid (AA). METHODS: Venous blood was taken from healthy human volunteers reported to be free of medications for one week. Platelet aggregation was monitored at 37 degree using Dual-channel Lumi-aggregometer. The resulting aggregation was recorded for 5 min by the measurement of light transmission as a function of time. RESULTS: The data show that a synergism in platelet aggregation mediated by subthreshold concentrations of epinephrine (1 micromol/L) and AA (0.2 micromol/L) was inhibited by the alpha2-receptor antagonist (yohimbine, IC50)=0.6 micromol/L) and an inhibitor of AA-cyclooxygenase (COX), indomethacin (IC50=0.25 micromol/L). In examining receptor influence on intraplatelet signalling pathways, it was found that the synergistic effect was inhibited by calcium channel blockers, verapamil (IC50=0.4 micromol/L) and diltiazem (IC50=2.5 micromol/L), as well as by low concentrations of inhibitors of phospholipase C (PLC) (U73122; IC50=0.2 micromol/L) and mitogens activated protein kinase (MAPK) (PD 98059; IC50=3.8 micromol/L). Herbimycin A, a specific inhibitor of tyrosine light chain kinase (TLCK), showed inhibition at IC50 value of 15 micromol/L, whereas chelerythrine, a protein kinase C (PKC) inhibitor, had no effect up to 20 micromol/L. CONCLUSION: These data suggest that synergism between epinephrine and AA in platelet aggregation is triggered through receptors coupled to G-protein, which in turn, activate PLC, COX, and MAP kinase-signaling pathways.