Topographical dissociation of BCL-2 messenger RNA and protein expression in human lymphoid tissues

Immunohistochemistry and in situ hybridization with a synthetic oligonucleotide probe were used to compare the topographical distribution of BCL-2 proto-oncogenic protein with that of its messenger RNA (mRNA) in normal lymphoid tissues, follicular lymphomas, and lymphoma-derived cell lines. In normal lymph nodes, BCL-2 protein was most abundant in the small lymphocytes of primary lymphoid follicles and the mantle zones of secondary follicles, virtually absent within germinal centers, and of variable abundance in many interfollicular cells. In contrast, the distribution of BCL-2 mRNA was roughly reciprocal to that of the protein with intense hybridization signal in germinal centers and almost none in mantle zones. Discordant BCL-2 RNA and protein levels were also observed in tonsillar epithelial cells and cortical thymocytes. Concordant and abundant expression of BCL-2 mRNA and protein was detected in biopsy tissues and cell lines from t(14;18)-carrying lymphomas. The contrasting distributions of BCL- 2 protein and RNA in normal lymphoid tissues suggest that translational and posttranslational control mechanisms play a significant role in regulating BCL-2 protein levels in germinal center cells, epithelial cells, and cortical thymocytes. Concordant BCL-2 mRNA and protein levels in follicular lymphomas suggest that translational control mechanisms may be disrupted as part of the sequence of genetic changes that transforms normal lymphoid cells into neoplastic follicular lymphoma cells.     

Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant

Mutations in polycystin-1 and transient receptor potential polycystin 2 (TRPP2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as a cation channel in its homomeric complex and in the TRPP2/polycystin-1 receptor/ion channel complex. The activation mechanism of TRPP2 is unknown, which significantly limits the study of its function and regulation. Here, we generated a constitutively active gain-of-function (GOF) mutant of TRPP2 by applying a mutagenesis scan on the S4–S5 linker and the S5 transmembrane domain, and studied functional properties of the GOF TRPP2 channel. We found that extracellular divalent ions, including Ca²⁺, inhibit the permeation of monovalent ions by directly blocking the TRPP2 channel pore. We also found that D643, a negatively charged amino acid in the pore, is crucial for channel permeability. By introducing single-point ADPKD pathogenic mutations into the GOF TRPP2, we showed that different mutations could have completely different effects on channel activity. The in vivo function of the GOF TRPP2 was investigated in zebrafish embryos. The results indicate that, compared with wild type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, including curly tail and cyst formation in the pronephric kidney, caused by down-regulation of endogenous TRPP2 expression. Thus, we established a GOF TRPP2 channel that can serve as a powerful tool for studying the function and regulation of TRPP2. The GOF channel may also have potential application for developing new therapeutic strategies for ADPKD.Autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases in humans, affects one in 400–1,000 individuals (1, 2). It is characterized by the formation of fluid-filled renal cysts that ultimately lead to kidney failure. ADPKD is caused by mutations in either the polycystin-1 or transient receptor potential polycystin 2 (TRPP2) protein (3). Polycystin-1, also known as PC1, a polycystic kidney disease (PKD) family member, is a large integral membrane protein with 11 transmembrane segments and a big extracellular N terminus (4). TRPP2, also known as polycystin-2 or PC2, is a member of the transient receptor potential (TRP) ion channel family, a large group of six-transmembrane cation channels that play crucial roles in sensory physiology (5, 6). Polycystin-1 and TRPP2 form a receptor/ion channel complex through the association of their C-terminal coiled-coil domains (7–10). Our previous work showed that this complex contains one polycystin-1 and three TRPP2 subunits (9, 10). How mutations in polycystin-1 or TRPP2 lead to ADPKD is unknown, but it is generally believed that the TRPP2/polycystin-1 complex functions as a sensor on primary cilia of renal epithelial cells to couple extracellular stimuli with intracellular Ca²⁺ signals (11–13). Presumably, disruption of this Ca²⁺ signaling leads to cyst formation in ADPKD. TRPP2 also forms a flow sensor with PKD1L1, another PKD protein, on the primary cilia of the embryonic nodal cells and plays a crucial role in establishing left/right asymmetry during early stages of development (14–17).TRPP2 is a Ca²⁺-permeable nonselective cation channel, and its channel function has been described both in the presence (12, 13, 18) and absence of polycystin-1 (19–21). TRPP2 is localized on multiple subcellular compartments, including the endoplasmic reticulum (ER) membrane, plasma membrane, and primary cilium, depending on the cell type or availability of binding partners (22). Correspondingly, TRPP2 appears to have distinct functions in each subcellular compartment (23, 24). For example, it functions as a calcium release channel on the ER membrane (21) and as a mechanosensitive channel in complex with polycystin-1 on the ciliary membrane (13). Due to the essential role of TRPP2 in ADPKD, understanding its channel function and regulation, and its role in renal physiology, are crucial. However, recording the TRPP2 current has been difficult, because TRPP2 is predominantly expressed in the ER instead of the plasma membrane (21, 25) and, more importantly, the activation mechanism of TRPP2 is still unknown. For this reason, almost all reported channel activity of TRPP2 is recorded at the single-channel level (19–21, 26–34), which has technical difficulties and limits the questions that can be addressed. The lack of a reliable system to record TRPP2 channel current easily has markedly delayed the study of this functionally important channel.A gain-of-function (GOF) TRPP2 can bypass the activation mechanism. Thus, it will facilitate the study of function and regulation of TRPP2 and help unravel the downstream signaling pathways. In this study, we took a systematic mutagenesis approach to obtain a constitutively active GOF mutant of the TRPP2 channel. GOF mutations of other TRP channels identified in diseases or in random mutagenesis studies indicated a common gating feature among these channels: mutations in the linker between the fourth and fifth transmembrane segments (S4–S5 linker) and the first half of the fifth transmembrane segment (S5) can lead to constitutive channel activity (35) (Fig. S1). In a previous study, proline (Pro)-scanning mutagenesis conducted in this region successfully generated GOF mutants of the TRPML1 channel (36). We applied a similar strategy to TRPP2 and found F604P, a mutation in the S5 segment, causing constitutive opening of the TRPP2 channel when expressed in Xenopus oocytes. This GOF TRPP2 opens up an avenue for studies on TRPP2, as partially shown in this report. Our results not only provide new insights into the function and regulation of TRPP2 but also indicate a potential use for the GOF TRPP2 in new therapeutic strategies for ADPKD treatment.Open in a separate windowFig. S1.Alignment of the fifth transmembrane segments of different TRP proteins showing the mutations in this region that lead to GOF. Red and underlined amino acids show the GOF mutation sites that were identified in disease or random mutagenesis studies. In addition to the F604P in TRPP2 (indicated with asterisk) that was found in this study, mutations shown in the figure are as follows: F550I in Drosophila TRPC1 channel (fTRPC) (84); T635A in mouse TRPC3 (85); M581T in rat TRPV1 (86); L619P and L623P in rat TRPV4 (87); R616Q and V620I in human TRPV4 (88); R427P, C430P, C431P, and V432P in mouse TRPML1 (36); A419P in mouse TRPML3 (89, 90); and F380L in yeast TRPY1 (91). Adapted from ref. (35), with the TRPP2 sequence added.     

Diet induced obesity in rats reduces NHE3 and Na+/K+‐ATPase expression in the kidney

The consumption of a high fat diet (HFD) is associated with proteinuria and altered sodium handling and excretion, which can lead to kidney disease. In the proximal tubule, the Na⁺/H⁺ Exchanger 3 (NHE3) is responsible for normal protein reabsorption and the reabsorption of approximately 70% of the renal sodium load. It is the Na⁺/K⁺‐ATPase that provides the driving force for the reabsorption of sodium and its exit across the basolateral membrane. This study investigates the effects that consumption of a HFD for 12 weeks has on NHE3 and Na⁺/K⁺‐ATPase expression in the kidney. Western blot analysis identified a significant reduction in NHE3 and its modulator, phosphorylated protein kinase B, in renal lysate from obese rats. In the obese rats, a reduction in NHE3 expression in the proximal tubule may impact on the acidification of endosomes which are responsible for albumin uptake, suggesting a key role for the exchanger in protein endocytosis in obesity. Western blot analysis identified a reduction in Na⁺/K⁺‐ATPase which could also potentially impact on albumin uptake and sodium reabsorption. This study demonstrates that consumption of a HFD for 12 weeks reduces renal NHE3 and Na⁺/K⁺‐ATPase expression, an effect that may contribute to the albuminuria associated with obesity. Furthermore the reduction in these transporters is not likely to contribute to the reduced sodium excretion in obesity. These data highlight a potential link between NHE3 and Na⁺/K⁺‐ATPase in the pathophysiological changes in renal protein handling observed in obesity.     

Repeated Social Stress Increases Reward Salience and Impairs Encoding of Prediction by Rat Locus Coeruleus Neurons

Stress is implicated in psychopathology characterized by cognitive dysfunction. Cognitive responses to stress are regulated by the locus coeruleus–norepinephrine (LC–NE) system. As social stress is a prevalent human stressor, this study determined the impact of repeated social stress on the relationship between LC neuronal activity and behavior during the performance of cognitive tasks. Social stress-exposed rats performed better at intradimensional set shifting (IDS) and made fewer perseverative errors during reversal learning (REV). LC neurons of control rats were task responsive, being activated after the choice and before reward. Social stress shifted LC neuronal activity from being task responsive to being reward responsive during IDS and REV. LC neurons of stressed rats were activated by reward and tonically inhibited by reward omission with incorrect choices. In contrast, LC neurons of stress-naive rats were only tonically inhibited by reward omission. Reward-related LC activation in stressed rats was unrelated to predictability because it did not habituate as learning progressed. The findings suggest that social stress history increases reward salience and impairs processes that compute predictability for LC neurons. These effects of social stress on LC neuronal activity could facilitate learning as indicated by improved performance in stressed rats. However, the ability of social stress history to enhance responses to behavioral outcomes may have a role in the association between stress and addictive behaviors. In addition, magnified fluctuations in LC activity in response to opposing behavioral consequences may underlie volatile changes in emotional arousal that characterize post-traumatic stress disorder.In response to acute stressors, neural systems that regulate the hypothalamic–pituitary–adrenal axis, autonomic function, behavior, and cognition are engaged in a coordinated manner to cope with the stressor and promote survival. Persistent or repeated activation of these stress systems as a result of chronic or repeated stress, or inappropriate activation of the systems in the absence of stress, is maladaptive and results in pathology (Chrousos and Gold, 1992; de Kloet et al, 2005; McEwen, 1998). Such maladaptive stress responses are thought to underlie symptoms such as hyperarousal, inappropriate fear, and attentional and cognitive dysfunctions that characterize certain psychiatric disorders (Gold and Chrousos, 2002). Interestingly, although many studies report impairment of cognitive processes by stressors, there are also reports of enhancement and these differences may depend on the specific stressor and the cognitive endpoint (Bondi et al, 2008; Butts et al, 2013; Graybeal et al, 2011; Lapiz-Bluhm et al, 2009; Thai et al, 2013). These differences underscore the complexity of effects of stress on neural circuits that regulate cognitive functions.The locus coeruleus–norepinephrine (LC–NE) system is a stress-response system that has been implicated in cognitive responses to stress and in stress-related psychopathology (Southwick et al, 1999; Valentino and Van Bockstaele, 2008; Wong et al, 2000). LC neurons are spontaneously active and their discharge rate is positively correlated to behavioral indices of arousal (Aston-Jones and Bloom, 1981b; Foote et al, 1980). Salient stimuli elicit a phasic activation of LC neurons that precedes orientation to the stimulus and this has implicated the system in attention (Aston-Jones and Bloom, 1981a; Berridge and Waterhouse, 2003; Foote et al, 1980). LC neuronal recordings in animals performing operant tasks indicate that phasic LC discharge is associated with focused attention and staying ‘on-task'', whereas high-tonic LC discharge is associated with labile attention and task disengagement (Aston-Jones and Cohen, 2005; Bouret and Sara, 2005; Sara, 2009). It has been suggested that this high-tonic mode of LC discharge facilitates cognitive flexibility (Aston-Jones and Cohen, 2005). Acute stressors and exposure to the stress-related peptide, corticotropin-releasing factor (CRF) bias LC activity towards a high-tonic state that may be adaptive in a dynamic environment with life-threatening challenges (Curtis et al, 2012; Valentino and Foote, 1987; Valentino and Wehby, 1988). Notably, certain doses of CRF improve extradimensional set-shifting performance during an attentional set-shifting task (AST), an endpoint of cognitive flexibility (Snyder et al, 2012). In contrast to the activating effects of acute stressors on LC neuronal activity, repeated social stress produces an enduring inhibition of rat LC discharge that is apparent several days after the last stressor (Chaijale et al, 2013). These findings are relevant to humans given that social stress is one of the most common human stressors. A similar enduring inhibition of LC activity occurs in rats exposed to a model of post-traumatic stress disorder that involves the administration of three consecutive stressors (George et al, 2013). It is unknown how this state of LC inhibition impacts on cognition.As cognitive dysfunction is thought to be one of the maladaptive consequences of repeated stress and the LC–NE system has been implicated in cognitive aspects of the stress response, the present study investigated the long-term effects of repeated social stress on LC activity recorded during the performance of an AST that assesses simple discrimination (SD), compound discrimination (CD), intradimensional set shifting (IDS), reversal learning (REV), and extradimensional set shifting. The results provided evidence that repeated social stress changes the relationship between LC neuronal activity and task performance, and renders LC neurons responsive to reward regardless of its predictability.     

Forebrain-Specific CRF Overproduction During Development is Sufficient to Induce Enduring Anxiety and Startle Abnormalities in Adult Mice

Corticotropin releasing factor (CRF) regulates physiological and behavioral responses to stress. Trauma in early life or adulthood is associated with increased CRF in the cerebrospinal fluid and heightened anxiety. Genetic variance in CRF receptors is linked to altered risk for stress disorders. Thus, both heritable differences and environmentally induced changes in CRF neurotransmission across the lifespan may modulate anxiety traits. To test the hypothesis that CRF hypersignaling is sufficient to modify anxiety-related phenotypes (avoidance, startle, and conditioned fear), we induced transient forebrain-specific overexpression of CRF (CRFOE) in mice (1) during development to model early-life stress, (2) in adulthood to model adult-onset stress, or (3) across the entire postnatal lifespan to model heritable increases in CRF signaling. The consequences of these manipulations on CRF peptide levels and behavioral responses were examined in adulthood. We found that transient CRFOE during development decreased startle habituation and prepulse inhibition, and increased avoidance (particularly in females) recapitulating the behavioral effects of lifetime CRFOE despite lower CRF peptide levels at testing. In contrast, CRFOE limited to adulthood reduced contextual fear learning in females and increased startle reactivity in males but did not change avoidance or startle plasticity. These findings suggest that forebrain CRFOE limited to development is sufficient to induce enduring alterations in startle plasticity and anxiety, while forebrain CRFOE during adulthood results in a different phenotype profile. These findings suggest that startle circuits are particularly sensitive to forebrain CRFOE, and that the impact of CRFOE may be dependent on the time of exposure.     

Healthcare resource utilization among haemophilia A patients in the United States

Summary. Advances in therapy have improved life expectancy and quality of life of patients with haemophilia A. Due to the chronic and complex management of this disease, particularly, the development of inhibitors, little is known about their health resource utilization in the real‐life setting over time. The aim was to assess the distribution and trend of healthcare resource utilization among US haemophilia A patients with and without inhibitors. The MarketScan® Database, was queried to identify individuals with ≥1 year continuous enrolment, two medical diagnoses of haemophilia A and claims for factor VIII or bypassing agent (to infer inhibitor status) during 2001–2007. Haemophilia‐related cost was estimated from inpatient, outpatient and pharmacy claims. Annual cost differences were assessed by age and over a 4‐year period for those with continuous enrolment. Among 51 million covered lives, 1044 haemophilia patients were identified, of whom 981 (94%; mean age = 21.2 years) did not have an inhibitor. The median haemophilia‐related cost for these patients was $63,935 per patient per year. When normalized by weight, annual cost was stable (no statistically significant differences) among 312 non‐inhibitor patients (mean age = 21.8 years) with 4‐year continuous data. While there was a wide distribution of haemophilia‐related cost among the 63 individuals with an inhibitor (mean age = 15.4 years), only 0.6% of the total haemophilia patients had costs exceeding $1 million per patient per year. This study indicated that most haemophilia A patients were inhibitor‐free with relatively stable annual costs over time. There was a wide distribution of haemophilia‐related cost for inhibitor patients, while the proportion of patients who incurred extreme high cost was low.