Coherent Rayleigh-Brillouin scattering measurements of bulk viscosity of polar and nonpolar gases, and kinetic theory.

We investigate coherent Rayleigh-Brillouin spectroscopy as an efficient process to measure the bulk viscosity of gases at gigahertz frequencies. Scattered spectral distributions are measured using a Fizeau spectrometer. We discuss the statistical error due to the fluctuating mode structure of the used pump laser. Experiments were done for both polar and nonpolar gases and the bulk viscosity was obtained from the spectra using the Tenti S6 model. Results are compared to simple classical kinetic models of molecules with internal degrees of freedom. At the extremely high (gigahertz) frequencies of our experiment, most internal vibrational modes remain frozen and the bulk viscosity is dominated by the rotational degrees of freedom. Our measurements show that the molecular dipole moments have unexpectedly little influence on the bulk viscosity at room temperature and moderate pressure.

Differential effect of alpha-syntrophin knockout on aquaporin-4 and Kir4.1 expression in retinal macroglial cells in mice.

Aquaporin-4 water channels and the inwardly rectifying potassium channels Kir4.1 are coexpressed in a highly polarized manner at the perivascular and subvitreal endfeet of retinal Müller cells and astrocytes. The present study was aimed at resolving the anchoring mechanisms responsible for the coexpression of these molecules. Both aquaporin-4 and Kir4.1 contain PDZ-domain binding motifs at their C-termini and it was recently shown that mice with targeted disruption of the dystrophin gene display altered distribution of aquaporin-4 and Kir4.1 in the retina. To test our hypothesis that alpha-syntrophin (a PDZ-domain containing protein of the dystrophin associated protein complex) is involved in aquaporin-4 and Kir4.1 anchoring in retinal cells, we studied the expression pattern of these molecules in alpha-syntrophin null mice. Judged by quantitative immunogold cytochemistry, deletion of the alpha-syntrophin gene causes a partial loss (by 70%) of aquaporin-4 labeling at astrocyte and Müller cell endfeet but no decrease in Kir4.1 labeling at these sites. These findings suggest that alpha-syntrophin is not involved in the anchoring of Kir4.1 and only partly responsible for the anchoring of aquaporin-4 in retinal endfeet membranes. Furthermore we show that wild type and alpha-syntrophin null mice exhibit strong beta1 syntrophin labeling at perivascular and subvitreal Müller cell endfeet, raising the possibility that beta1 syntrophin might be involved in the anchoring of Kir4.1 and the alpha-syntrophin independent pool of aquaporin-4.

Alternative transcripts and evidence of imprinting of GNAL on 18p11.2.

Genetic studies implicating the region of human chromosome 18p11.2 in susceptibility to bipolar disorder and schizophrenia have observed parent-of-origin effects that may be explained by genomic imprinting. We have identified a transcriptional variant of the GNAL gene in this region, employing an alternative first exon that is 5' to the originally identified start site. This alternative GNAL transcript encodes a longer functional variant of the stimulatory G-protein alpha subunit, Golf. The isoforms of Golf display different expression patterns in the CNS and functionally couple to the dopamine D1 receptor when heterologously expressed in Sf9 cells. In addition, there are CpG islands in the vicinity of both first exons that are differentially methylated, a hallmark of genomic imprinting. These results suggest that GNAL, and possibly other genes in the region, is subject to epigenetic regulation and strengthen the case for a susceptibility gene in this region.

Deficiency of the syntrophins and alpha-dystrobrevin in patients with inherited myopathy.

The syntrophins and dystrobrevins are members of the dystrophin-associated protein complex, and are thought to function as modular adaptors for signalling proteins recruited to the sarcolemmal membrane. We have characterised the expression of the syntrophins (alpha-, beta1-, and beta2-) and alpha-dystrobrevin by immunohistochemistry in normal human muscle and in biopsies from 162 patients with myopathies of unknown aetiology (with normal staining for dystrophin and other dystrophin-associated proteins). Unlike mice, beta2-syntrophin is expressed at the sarcolemma in post-natal human skeletal muscle. Deficiency of alpha-dystrobrevin +/- beta2-syntrophin was present in 16/162 (10%) patients, compared to age-matched controls. All patients presented with congenital-onset hypotonia and weakness, although there was variability in clinical severity. Two major clinical patterns emerged: patients with deficiency of beta2-syntrophin and alpha-dystrobrevin presented with severe congenital weakness and died in the first year of life, and two patients with deficiency of alpha-dystrobrevin had congenital muscular dystrophy with complete external ophthalmoplegia. We have sequenced the coding regions of alpha-dystrobrevin and beta2-syntrophin in these patients, and identified a new isoform of dystrobrevin, but have not identified any mutations. This suggests that disease causing mutations occur outside the coding region of these genes, in gene(s) encoding other components of the syntrophin-dystrobrevin subcomplex, or in gene(s) responsible for their post-translational modification and normal localisation.

Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity.

Expanded polyglutamine repeats have been proposed to cause neuronal degeneration in Huntington's disease (HD) and related disorders, through abnormal interactions with other proteins containing short polyglutamine tracts such as the transcriptional coactivator CREB binding protein, CBP. We found that CBP was depleted from its normal nuclear location and was present in polyglutamine aggregates in HD cell culture models, HD transgenic mice, and human HD postmortem brain. Expanded polyglutamine repeats specifically interfere with CBP-activated gene transcription, and overexpression of CBP rescued polyglutamine-induced neuronal toxicity. Thus, polyglutamine-mediated interference with CBP-regulated gene transcription may constitute a genetic gain of function, underlying the pathogenesis of polyglutamine disorders.

Isolation of a 40-kDa Huntingtin-associated protein.

Huntington's disease is caused by an expanded CAG trinucleotide repeat coding for a polyglutamine stretch within the huntingtin protein. Currently, the function of normal huntingtin and the mechanism by which expanded huntingtin causes selective neurotoxicity remain unknown. Clues may come from the identification of huntingtin-associated proteins (HAPs). Here, we show that huntingtin copurifies with a single novel 40-kDa protein termed HAP40. HAP40 is encoded by the open reading frame factor VIII-associated gene A (F8A) located within intron 22 of the factor VIII gene. In transfected cell extracts, HAP40 coimmunoprecipitates with full-length huntingtin but not with an N-terminal huntingtin fragment. Recombinant HAP40 is cytoplasmic in the presence of huntingtin but is actively targeted to the nucleus in the absence of huntingtin. These data indicate that HAP40 is likely to contribute to the function of normal huntingtin and is a candidate for involvement in the aberrant nuclear localization of mutant huntingtin found in degenerating neurons in Huntington's disease.

Brazilian family with pure autosomal dominant spastic paraplegia maps to 8q: analysis of muscle beta 1 syntrophin.

The autosomal dominant hereditary spastic paraplegias (AD-HSP) are a heterogeneous group of degenerative disorders of the central motor system, characterized by progressive spasticity of the lower limbs. Five loci for pure AD-HSP have been identified to date: SPG3 at 14q, SPG4 at 2p, SPG6 at 15q, SPG8 at 8q, and more recently SPG10 at 12q. We have analyzed a Brazilian family with 16 affected individuals by pure AD-HSP who developed progressive gait disturbance with onset at age 18-26 years. Linkage analysis performed with 13 relatives (6 affected and 7 normal) excluded SPG3, SPG4, and SPG6 as candidate regions. However, positive LOD scores were obtained with markers flanking the candidate region for the SPG8 locus [maximum two point Lod score (Zmax) = 3.3 at theta = 0 for D8S1804]. In this region lies the syntrophin beta 1 gene (SNT2B1), a widely expressed dystrophin-associated protein and therefore a good positional and functional candidate for this disease. Immunohistochemical and Western Blot (WB) studies showed that the distribution, expression, and apparent molecular weight of the beta 1 syntrophin protein were comparable to those of normal control individuals. Therefore, it is unlikely that defects in this protein are related to SPG8, at least in the present family.

Preparation of human cDNas encoding expanded polyglutamine repeats.

At least eight neurodegenerative diseases result from expansions of polyglutamine tracts encoded by CAG trinucleotide repeats. Although polyglutamine diseases typically have onset after age 50 in humans, these diseases can be modeled in animals and in cell culture by using highly expanded repeats to accelerate the pathogenesis. Unfortunately, current methods for preparing recombinant constructs with large glutamine tracts either alter the coding region adjacent to the repeat or yield highly unstable pure CAG repeats. We have developed a technique for expanding repeats that results in a more stable mix of CAG and CAA glutamine codons. We expect this technique to allow rapid preparation of highly expand repeats suitable for stable animal and cell culture models for any of the polyglutamine repeat diseases.

Nuclear targeting of mutant Huntingtin increases toxicity.

Huntington's disease is a neurodegenerative disorder resulting from expansion of the polyglutamine region in huntingtin. Although huntingtin is normally cytoplasmic, in affected brain regions proteolytic fragments of mutant huntingtin containing the polyglutamine repeat form intranuclear inclusions. Here, we examine the contribution of nuclear localization to toxicity by transiently transfecting neuro-2a cells with an N-terminal huntingtin fragment similar in size to that believed to be present in patients. The huntingtin fragment, HD-N63, was targeted either to the cytoplasm with a nuclear export signal (NES) or to the nucleus with a nuclear localization signal (NLS). The NES decreased the number of cells with aggregates in the nucleus while an NLS had the opposite effect. By cotransfecting HD-N63 with GFP as a marker, we observed direct cell loss with constructs containing expanded polyglutamine repeats. Compared to unmodified HD-N63-75Q, adding an NES reduced cell loss by 57% while an NLS increased cell loss by 111%. These results indicate that nuclear localization of mutant huntingtin fragments plays an important role in cell toxicity.

Polyglutamine pathogenesis.

An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis.

Differential membrane localization and intermolecular associations of alpha-dystrobrevin isoforms in skeletal muscle.

alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin. alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins.

Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanding CAG repeat coding for polyglutamine in the huntingtin protein. Recent data have suggested the possibility that an N-terminal fragment of huntingtin may aggregate in neurons of patients with HD, both in the cytoplasm, forming dystrophic neurites, and in the nucleus, forming intranuclear neuronal inclusion bodies. An animal model of HD using the short N-terminal fragment of huntingtin has also been found to have intranuclear inclusions and this same fragment can aggregate in vitro . We have now developed a cell culture model demonstrating that N-terminal fragments of huntingtin with expanded glutamine repeats aggregate both in the cytoplasm and in the nucleus. Neuroblastoma cells transiently transfected with full-length huntingtin constructs with either a normal or expanded repeat had diffuse cytoplasmic localization of the protein. In contrast, cells transfected with truncated N-terminal fragments showed aggregation only if the glutamine repeat was expanded. The aggregates were often ubiquitinated. The shorter truncated product appeared to form more aggregates in the nucleus. Cells transfected with the expanded repeat construct but not the normal repeat construct showed enhanced toxicity to the apoptosis-inducing agent staurosporine. These data indicate that N-terminal truncated fragments of huntingtin with expanded glutamine repeats can aggregate in cells in culture and that this aggregation can be toxic to cells. This model will be useful for future experiments to test mechanisms of aggregation and toxicity and potentially for testing experimental therapeutic interventions.

Differential association of syntrophin pairs with the dystrophin complex.

The syntrophins are a multigene family of intracellular dystrophin-associated proteins comprising three isoforms, alpha1, beta1, and beta2. Based on their domain organization and association with neuronal nitric oxide synthase, syntrophins are thought to function as modular adapters that recruit signaling proteins to the membrane via association with the dystrophin complex. Using sequences derived from a new mouse beta1-syntrophin cDNA, and previously isolated cDNAs for alpha1- and beta2-syntrophins, we prepared isoform-specific antibodies to study the expression, skeletal muscle localization, and dystrophin family association of all three syntrophins. Most tissues express multiple syntrophin isoforms. In mouse gastrocnemius skeletal muscle, alpha1- and beta1-syntrophin are concentrated at the neuromuscular junction but are also present on the extrasynaptic sarcolemma. beta1-syntrophin is restricted to fast-twitch muscle fibers, the first fibers to degenerate in Duchenne muscular dystrophy. beta2-syntrophin is largely restricted to the neuromuscular junction. The sarcolemmal distribution of alpha1- and beta1-syntrophins suggests association with dystrophin and dystrobrevin, whereas all three syntrophins could potentially associate with utrophin at the neuromuscular junction. Utrophin complexes immunoisolated from skeletal muscle are highly enriched in beta1- and beta2-syntrophins, while dystrophin complexes contain mostly alpha1- and beta1-syntrophins. Dystrobrevin complexes contain dystrophin and alpha1- and beta1-syntrophins. From these results, we propose a model in which a dystrophin-dystrobrevin complex is associated with two syntrophins. Since individual syntrophins do not have intrinsic binding specificity for dystrophin, dystrobrevin, or utrophin, the observed preferential pairing of syntrophins must depend on extrinsic regulatory mechanisms.

Purification, cDNA sequence, and tissue distribution of rat uroguanylin.

Guanylin, a peptide purified from rat jejunum, is thought to regulate water and electrolyte balance in the intestine. We show here, using a combination of Northern blots, Western blots, and functional assays, that guanylin and its receptor (GCC) are not distributed in parallel within the rat intestine. To investigate the possibility that there might be a second intestinal peptide that serves as a ligand for GCC, we assayed tissue extracts for the ability to stimulate cyclic GMP synthesis in a GCC-expression cell line. Duodenal extracts display a peak of biological activity that is not present in colon and that does not comigrate with guanylin or proguanylin. The activity co-purifies with a novel peptide (TIATDECELCINVACTGC) that has high homology with uroguanylin, a peptide initially purified from human and opossum urine. A rat uroguanylin cDNA clone was found to encode a propeptide whose C-terminus corresponds to our purified peptide. Northern blots with probes generated from this clone reveal that prouroguanylin mRNA is strongly expressed in proximal small intestine, but virtually absent from colon, corroborating our biochemical measurements. Taken together, these studies demonstrate an intestinal origin for uroguanylin, and show that within the intestine its distribution is complementary to that of guanylin.

beta-dystrobrevin, a new member of the dystrophin family. Identification, cloning, and protein associations.

Dystrophin, the protein disrupted in Duchenne muscular dystrophy, is one of several related proteins that are key components of the submembrane cytoskeleton. Three dystrophin-related proteins (utrophin, dystrophin-related protein-2 (DRP2), and dystrobrevin) have been described. Here, we identify a human gene on chromosome 2p22-23 that encodes a novel protein, beta-dystrobrevin, with significant homology to the other known dystrobrevin (now termed alpha-dystrobrevin). Sequence alignments including this second dystrobrevin strongly support the concept that two distinct subfamilies exist within the dystrophin family, one composed of dystrophin, utrophin, and DRP2 and the other composed of alpha- and beta-dystrobrevin. The possibility that members of each subfamily form distinct protein complexes was examined by immunopurifying dystrobrevins and dystrophin. A beta-dystrobrevin antibody recognized a protein of the predicted size (71 kDa) that copurified with the dystrophin short form, Dp71. Thus, like alpha-dystrobrevin, beta-dystrobrevin is likely to associate directly with dystrophin. alpha- and beta-dystrobrevins failed to copurify with each other, however. These results suggest that members of the dystrobrevin subfamily form heterotypic associations with dystrophin and raise the possibility that pairing of a particular dystrobrevin with dystrophin may be regulated, thereby providing a mechanism for assembly of distinct submembrane protein complexes.

Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains.

Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

Isoform diversity of dystrobrevin, the murine 87-kDa postsynaptic protein.

Dystrophin-related and -associated proteins are important in the formation and maintenance of the mammalian neuromuscular junction. We have characterized mouse cDNA clones encoding isoforms of the dystrophin-homologous 87-kDa postsynaptic protein, dystrobrevin. In Torpedo, the 87-kDa protein is multiply phosphorylated and closely associated with proteins in the postsynaptic cytoskeleton, including the acetylcholine receptor. In contrast to Torpedo, where only a single transcript is seen, the mouse expresses several mRNAs encoding different isoforms. A 6.0-kilobase transcript in brain encodes a 78-kDa protein (dystrobrevin-2) that has a different C terminus, lacking the putative tyrosine kinase substrate domain. In skeletal and cardiac muscle, transcripts of 1.7 and 3.3/3.5 kilobases predominate and encode additional isoforms. Alternative splicing within the coding region and differential usage of untranslated regions produce additional variation. Multiple dystrobrevin-immunoreactive proteins copurify with syntrophin from mouse tissues. In skeletal muscle, dystrobrevin immunoreactivity is restricted to the neuromuscular junction and sarcolemma. The occurrence of many dystrobrevin isoforms is significant because alternative splicing and phosphorylation often have profound effects upon the biological activity of synaptic proteins.

beta 2-Syntrophin: localization at the neuromuscular junction in skeletal muscle.

The syntrophins are a multigene family of proteins which bind C-terminal domains of dystrophin, utrophin and homologs thereof. We report here that antibodies specific for one isoform, beta 2-syntrophin, labeled only the neuromuscular junction (NMJ) in rat skeletal muscle. Anti-alpha 1-syntrophin antibodies gave strong labeling of the sarcolemma and NMJ in normal rat and mouse muscle, and similar but much weaker labeling in dystrophin-minus mdx muscle. beta 2-Syntrophin therefore appears to be specific to the NMJ in normal muscle, as is utrophin, and may be involved in acetylcholine receptor clustering. alpha 1-Syntrophin appears to be associated mainly with dystrophin, as expected, but a small portion must be associated with another protein, possibly homologs of the electric tissue 87K protein.