Gordon Holmes syndrome (GHS) is a rare Mendelian neurodegenerative disorder characterized by ataxia and hypogonadism. Recently, it was suggested that disordered ubiquitination underlies GHS though the discovery of exome mutations in the E3 ligase RNF216 and deubiquitinase OTUD4. We performed exome sequencing in a family with two of three siblings afflicted with ataxia and hypogonadism and identified a homozygous mutation in STUB1 (NM_005861) c.737C→T, p.Thr246Met, a gene that encodes the protein CHIP (C-terminus of HSC70-interacting protein). CHIP plays a central role in regulating protein quality control, in part through its ability to function as an E3 ligase. Loss of CHIP function has long been associated with protein misfolding and aggregation in several genetic mouse models of neurodegenerative disorders; however, a role for CHIP in human neurological disease has yet to be identified. Introduction of the Thr246Met mutation into CHIP results in a loss of ubiquitin ligase activity measured directly using recombinant proteins as well as in cell culture models. Loss of CHIP function in mice resulted in behavioral and reproductive impairments that mimic human ataxia and hypogonadism. We conclude that GHS can be caused by a loss-of-function mutation in CHIP. Our findings further highlight the role of disordered ubiquitination and protein quality control in the pathogenesis of neurodegenerative disease and demonstrate the utility of combining whole-exome sequencing with molecular analyses and animal models to define causal disease polymorphisms.

During the course of biological aging, there is a gradual accumulation of damaged proteins and a concomitant functional decline in the protein degradation system. Protein quality control is normally ensured by the coordinated actions of molecular chaperones and the protein degradation system that collectively help to maintain protein homeostasis. The carboxyl terminus of Hsp70-interacting protein (CHIP), a ubiquitin ligase/ cochaperone, participates in protein quality control by targeting a broad range of chaperone substrates for proteasome degradation via the ubiquitin-proteasome system, demonstrating a broad involvement of CHIP in maintaining cytoplasmic protein quality control. In the present study, we have investigated the influence that protein quality control exerts on the aging process by using CHIP ؊/؊ mice. CHIP deficiency in mice leads to a markedly reduced life span, along with accelerated age-related pathophysiological phenotypes. These features were accompanied by indications of accelerated cellular senescence and increased indices of oxidative stress. In addition, CHIP ؊/؊ mice exhibit a deregulation of protein quality control, as indicated by elevated levels of toxic oligomer proteins and a decline in proteasome activity. Taken together, these data reveal that impaired protein quality control contributes to cellular senescence and implicates CHIP-dependent quality control mechanisms in the regulation of mammalian longevity in vivo.

Understanding the physiological role of tachykinins requires precise cellular and subcellular localization of their receptors. We raised antisera by immunizing rabbits with peptides corresponding to portions of the intracellular tails of the rat neurokinin 1, 2, and 3 receptors (NK1-R, NK2-R, NK3-R). Receptors were localized by immunofluorescence and confocal microscopy. NK1-R, NK2-R, and NK3-R were detected at the plasma membrane of transfected cells with minimal intracellular stores. Staining was abolished by preabsorption of the antisera with the peptides used for immunization. Nontransfected cells were unstained. Each antiserum only stained cells transfected with the appropriate receptor and did not stain cells transfected with the other receptors. Therefore, the antisera are specific and do not cross-react with other neurokinin receptors. We examined the distribution of the neurokinin receptors in the gastrointestinal tract of the rat. NK1-R was detected in myenteric and submucosal neurons and in interstitial cells of Cajal. NK2-R was localized to circular and longitudinal muscle cells and to nerve endings in the plexuses. NK3-R was detected in numerous myenteric and submucosal neurons. Some neurons expressed both NK1-R and NK3-R. Receptors were detected at the plasma membrane and in endosomes. Cells expressing the receptors were closely associated with tachykinin-containing nerve fibers. Thus, NK1-R and NK3-R mediate neurotransmission by tachykinins within enteric nerve plexuses, and NK1-R and NK2-R mediate the effects of tachykinins on interstitial and smooth muscle cells, respectively.

Tachykinins, including substance P, neurokinin A, and neuropeptides K and gama, are expressed widely in the peripheral nervous system where they affect smooth muscle contraction, exocrine gland secretion, vascular permeability, and neurotransmission. Substance P, the preferred ligand for the NK1 receptor, is found in high concentrations in the enteric nervous system. In the present study, the localisation and distribution of the NK1 receptor was studied throughout the gastrointestinal tract of the guinea-pig by using a polyclonal antiserum raised against the C-terminal 15 amino acids of the NK1 receptor. Co-localisation with other neuronal markers was examined in the ileum. Nerve cell bodies reactive for the NK1 receptor were found in the myenteric plexus of all regions and the submucous plexus of the small and large intestines. In the small intestine, the interstitial cells of Cajal were also immunoreactive. Immunoreactivity was largely confined to cell surfaces. Almost all immunoreactive myenteric nerve cells had Dogiel type I morphology, and most of these were immunoreactive for nitric oxide synthase, a transmitter of inhibitory neurons to the muscle and of descending interneurons. Neuropeptide Y-containing secretomotor neurons in the submucous and myenteric plexuses also exhibited NK1 receptor immunoreactivity. NK1 receptors were present on a minority of tachykinin immunoreactive neurons of submucous ganglia. The results suggest that receptors on the longitudinal muscle might not be conventional NK1 receptors, that excitation of the circular muscle of the ileum is indirect, perhaps via the interstitial cells of Cajal, and that enteric inhibitory neurons may be excited via NK1 receptors.

The assembly and maintenance of the cardiac sarcomere, which contains the basic contractile components of actin and myosin, are essential for cardiac function. While often described as a static structure, the sarcomere is actually dynamic and undergoes constant turnover, allowing it to adapt to physiological changes while still maintaining function. A host of new factors have been identified that play a role in the regulation of protein quality control in the sarcomere, including chaperones that mediate the assembly of sarcomere components and ubiquitin ligases that control their specific degradation. There is clear evidence of sarcomere disorganization in animal models lacking muscle-specific chaperone proteins, illustrating the importance of these molecules in sarcomere structure and function. Although ubiquitin ligases have been found within the sarcomere structure itself, the role of the ubiquitin proteasome system in cardiac sarcomere regulation, and the factors that control its activity, are only just now being elucidated. The number of ubiquitin ligases identified with specificity for sarcomere proteins, each with distinct target substrates, is growing, allowing for tight regulation of this system. In this review, we highlight the dynamic interplay between sarcomere-specific chaperones and ubiquitin-dependent degradation of sarcomere proteins that is necessary in order to maintain structure and function of the cardiac sarcomere.

Rationale Among the extracellular modulators of Bmp (bone morphogenetic protein) signaling, Bmper (Bmp endothelial cell precursor-derived regulator) both enhances and inhibits Bmp signaling. Recently we found that Bmper modulates Bmp4 activity via a concentration-dependent, endocytic trap-and-sink mechanism. Objective To investigate the molecular mechanisms required for endocytosis of the Bmper/Bmp4 and signaling complex and determine the mechanism of Bmper’s differential effects on Bmp4 signaling. Methods and Results Using an array of biochemical and cell biology techniques, we report that LRP1 (Low density lipoprotein receptor-related protein 1), a member of the LDL receptor family, acts as an endocytic receptor for Bmper and a co-receptor of Bmp4 to mediate the endocytosis of the Bmper/Bmp4 signaling complex. Furthermore, we demonstrate that LRP1-dependent Bmper/Bmp4 endocytosis is essential for Bmp4 signaling, as evidenced by the phenotype of lrp1-deficient zebrafish, which have abnormal cardiovascular development and decreased Smad1/5/8 activity in key vasculogenic structures. Conclusions Together, these data reveal a novel role for LRP1 in the regulation of Bmp4 signaling by regulating receptor complex endocytosis. In addition, these data introduce LRP1 as a critical regulator of vascular development. These observations demonstrate Bmper’s ability to fine-tune Bmp4 signaling at the single-cell level, unlike the spatial regulatory mechanisms applied by other Bmp modulators.

The distribution of neurokinin-2 (NK2) tachykinin receptors was investigated by immunohistochemistry in the guinea-pig oesophagus, stomach, small and large intestine. Receptor immunoreactivity occurred at the surfaces of smooth muscle cells throughout the digestive tract. Nerve fibre varicosities in enteric ganglia were also immunoreactive. In myenteric ganglia, these varicosities were most numerous in the ileum, frequent, but less dense, in the proximal colon and caecum, rare in the distal colon, extremely infrequent in the rectum and duodenum, and absent from the stomach and oesophagus. Reactive varicosities were rare in the submucous ganglia. Reactive nerve fibres in the mucosa were only found in the caecum and proximal colon. Strong NK2 receptor immunoreactivity was also found on the surfaces of enterocytes at the bases of mucosal glands in the proximal colon. Receptors were not detectable on the surfaces of nerve cells or on non-terminal axons. Reactivity did not occur on nerve fibres innervating the muscle. Denervation studies showed that the immunoreactive varicosities in the myenteric plexus of the ileum were the terminals of descending interneurons. Immunoreactivity for nitric oxide synthase was colocalised with NK2 receptor (NK-R) immunoreactivity in about 70% of the myenteric varicosities in the small intestine. Bombesin immunoreactivity occurred in about 30% of NK2-R immunoreactive varicosities in the small intestine.

. Catecholamines act via a ␤-adrenergic receptor to maintain fetal heart rate and survival. Am J Physiol Heart Circ Physiol 284: H2069-H2077, 2003. First published February 6, 2003 10.1152/ajpheart.00588.2002Mice lacking catecholamines die before birth, some with cardiovascular abnormalities. To investigate the role of catecholamines in development, embryonic day 12.5 (E12.5) fetuses were cultured and heart rate monitored. Under optimal oxygenation, wild-type and catecholamine-deficient fetuses had the same initial heart rate (200-220 beats/min), which decreased by 15% in wild-type fetuses during 50 min of culture. During the same culture period, catecholamine-deficient fetuses dropped their heart rate by 35%. Hypoxia reduced heart rate of wild-type fetuses by 35-40% in culture and by 20% in utero, assessed by echocardiography. However, catecholamine-deficient fetuses exhibited greater hypoxia-induced bradycardia, reducing their heart rate by 70-75% in culture. Isoproterenol, a ␤-adrenergic receptor (␤-AR) agonist, reversed this extreme bradycardia, restoring the rate of catecholamine-deficient fetuses to that of nonmutant siblings. Moreover, isoproterenol rescued 100% of catecholamine-deficient pups to birth in a dose-dependent, stereospecific manner when administered in the dam's drinking water. An ␣-AR agonist was without effect. When wild-type fetuses were cultured with adrenoreceptor antagonists to create pharmacological nulls, blockade of ␣-ARs with 10 M phentolamine or ␤-ARs with 10 M bupranolol alone or in combination did not reduce heart rate under optimal oxygenation. However, when combined with hypoxia, ␤-AR blockade reduced heart rate by 35%. In contrast, the muscarinic blocker atropine and the ␣-AR antagonist phentolamine had no effect. These data suggest that ␤-ARs mediate survival in vivo and regulate heart rate in culture. We hypothesize that norepinephrine, acting through ␤-ARs, maintains fetal heart rate during periods of transient hypoxia that occur throughout gestation, and that catecholamine-deficient fetuses die because they cannot withstand hypoxia-induced bradycardia. tyrosine hydroxylase; dopamine ␤-hydroxylase; norepinephrine; hypoxia; adrenoreceptor DESPITE THE FACT THAT IN RODENTS catecholamines and their adrenergic receptors (AR) are present as early as embryonic day 8 and 9 (E8-9) (4, 33), pharmacological experiments suggested that ␤-AR activation by norepinephrine and epinephrine was not essential in the fetus. Pregnant rat dams treated with a nonselective