Raising cGMP restores proteasome function and myelination in mice with a proteotoxic neuropathy.

Agents that raise cyclic guanosine monophosphate (cGMP) by activating protein kinase G increase 26S proteasome activities, protein ubiquitination and degradation of misfolded proteins. Therefore, they may be useful in treating neurodegenerative and other diseases caused by an accumulation of misfolded proteins. Mutations in myelin protein zero (MPZ) cause the peripheral neuropathy Charcot-Marie-Tooth type 1B (CMT1B). In peripheral nerves of a mouse model of CMT1B, where the mutant MPZS63del is expressed, proteasome activities are reduced, mutant MPZS63del and polyubiquitinated proteins accumulate and the unfolded protein response (p-eif2α) is induced. In HEK293 cells, raising cGMP stimulated ubiquitination and degradation of MPZS63del, but not of wild-type MPZ. Treating S63del mice with the phosphodiesterase 5 inhibitor, sildenafil-to raise cGMP-increased proteasome activity in sciatic nerves and reduced the levels of polyubiquitinated proteins, the proteasome reporter ubG76V-GFP and p-elF2α. Furthermore, sildenafil treatment reduced the number of amyelinated axons, and increased myelin thickness and nerve conduction velocity in sciatic nerves. Thus, agents that raise cGMP, including those widely used in medicine, may be useful therapies for CMT1B and other proteotoxic diseases.

cGMP via PKG activates 26S proteasomes and enhances degradation of proteins, including ones that cause neurodegenerative diseases.

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington's disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.

Corrigendum: YAP and TAZ Regulate Cc2d1b and Purß in Schwann Cells.

[This corrects the article DOI: 10.3389/fnmol.2019.00177.].

26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation.

Pharmacological agents that raise cAMP and activate protein kinase A (PKA) stimulate 26S proteasome activity, phosphorylation of subunit Rpn6, and intracellular degradation of misfolded proteins. We investigated whether a similar proteasome activation occurs in response to hormones and under various physiological conditions that raise cAMP. Treatment of mouse hepatocytes with glucagon, epinephrine, or forskolin stimulated Rpn6 phosphorylation and the 26S proteasomes' capacity to degrade ubiquitinated proteins and peptides. These agents promoted the selective degradation of short-lived proteins, which are misfolded and regulatory proteins, but not the bulk of cell proteins or lysosomal proteolysis. Proteasome activities and Rpn6 phosphorylation increased similarly in working hearts upon epinephrine treatment, in skeletal muscles of exercising humans, and in electrically stimulated rat muscles. In WT mouse kidney cells, but not in cells lacking PKA, treatment with antidiuretic hormone (vasopressin) stimulated within 5-minutes proteasomal activity, Rpn6 phosphorylation, and the selective degradation of short-lived cell proteins. In livers and muscles of mice fasted for 12-48 hours cAMP levels, Rpn6 phosphorylation, and proteasomal activities increased without any change in proteasomal content. Thus, in vivo cAMP-PKA-mediated proteasome activation is a common cellular response to diverse endocrine stimuli and rapidly enhances the capacity of target tissues to degrade regulatory and misfolded proteins (e.g., proteins damaged upon exercise). The increased destruction of preexistent regulatory proteins may help cells adapt their protein composition to new physiological conditions.

Exploring the Regulation of Proteasome Function by Subunit Phosphorylation.

Rates of degradation by the ubiquitin proteasome system depend not only on rates of ubiquitination, but also on the level of proteasome activity which can be regulated through phosphorylation of proteasome subunits. Many protein kinases have been proposed to influence proteasomal activity. However, for only two is there strong evidence that phosphorylation of a specific 26S subunit enhances the proteasome's capacity to degrade ubiquitinated proteins and promotes protein breakdown in cells: (1) protein kinase A (PKA), which after a rise in cAMP phosphorylates the 19S subunit Rpn6, and (2) dual tyrosine receptor kinase 2 (DYRK2), which during S through M phases of the cell cycle phosphorylates the 19S ATPase subunit Rpt3. In this chapter, we review and discuss the different methods used to assess the impact of phosphorylation by these two kinases on proteasomal activity and intracellular protein degradation. In addition, we present one method to determine if phosphorylation is responsible for an observed increase in proteasomal activity and another to evaluate by Phos-tag gel electrophoresis whether a specific proteasome subunit is modified by phosphorylation. The methods reviewed and presented here should be useful in clarifying the roles of other kinases and other posttranslational modifications of proteasome subunits.

Impairment of protein degradation and proteasome function in hereditary neuropathies.

In several neurodegenerative diseases in which misfolded proteins accumulate there is impairment of the ubiquitin proteasome system (UPS). We tested if a similar disruption of proteostasis occurs in hereditary peripheral neuropathies. In sciatic nerves from mouse models of two human neuropathies, Myelin Protein Zero mutation (S63del) and increased copy number (P0 overexpression), polyubiquitinated proteins accumulated, and the overall rates of protein degradation were decreased. 26S proteasomes affinity-purified from sciatic nerves of S63del mice were defective in degradation of peptides and a ubiquitinated protein, unlike proteasomes from P0 overexpression, which appeared normal. Nevertheless, cellular levels of 26S proteasomes were increased in both, through the proteolytic-activation of the transcription factor Nrf1, as occurs in response to proteasome inhibitors. In S63del, increased amounts of the deubiquitinating enzymes USP14, UCH37, and USP5 were associated with proteasomes, the first time this has been reported in a human disease model. Inhibitors of USP14 increased the rate of protein degradation in S63del sciatic nerves and unexpectedly increased the phosphorylation of eIF2α by Perk. Thus, proteasome content, composition and activity are altered in these diseases and USP14 inhibitors have therapeutic potential in S63del neuropathy.

Regulating protein breakdown through proteasome phosphorylation.

The ubiquitin proteasome system degrades the great majority of proteins in mammalian cells. Countless studies have described how ubiquitination promotes the selective degradation of different cell proteins. However, there is a small but the growing literature that protein half-lives can also be regulated by post-translational modifications of the 26S proteasome. The present study reviews the ability of several kinases to alter proteasome function through subunit phosphorylation. For example, PKA (protein kinase A) and DYRK2 (dual-specificity tyrosine-regulated kinase 2) stimulate the proteasome's ability to degrade ubiquitinated proteins, peptides, and adenosine triphosphate, while one kinase, ASK1 (apoptosis signal-regulating kinase 1), inhibits proteasome function during apoptosis. Proteasome phosphorylation is likely to be important in regulating protein degradation because it occurs downstream from many hormones and neurotransmitters, in conditions that raise cyclic adenosine monophosphate or cyclic guanosine monophosphate levels, after calcium influx following synaptic depolarization, and during phases of the cell cycle. Beyond its physiological importance, pharmacological manipulation of proteasome phosphorylation has the potential to combat various diseases. Inhibitors of phosphodiesterases by activating PKA or PKG (protein kinase G) can stimulate proteasomal degradation of misfolded proteins that cause neurodegenerative or myocardial diseases and even reduce the associated pathology in mouse models. These observations are promising since in many proteotoxic diseases, aggregation-prone proteins impair proteasome function, and disrupt protein homeostasis. Conversely, preventing subunit phosphorylation by DYRK2 slows cell cycle progression and tumor growth. However, further research is essential to determine how phosphorylation of different subunits by these (or other) kinases alters the properties of this complex molecular machine and thus influence protein degradation rates.

The role of perception in the mislocalization of the final position of a moving target.

The judged final position of a moving stimulus has been suggested to be shifted in the direction of motion because of mental extrapolation (representational momentum). However, a perceptual explanation is possible: The eyes overshoot the final position of the target, and because of a foveal bias, the judged position is shifted in the direction of motion. To test this hypothesis, the authors replicated previous studies, but instead of having participants indicate where the target vanished, the authors probed participants' perceptual focus by presenting probe stimuli close to the vanishing point. Identification of probes in the direction of target motion was more accurate immediately after target offset than it was with a delay. Another experiment demonstrated that judgments of the final position of a moving target are affected by whether the eyes maintain fixation or follow the target. The results are more consistent with a perceptual explanation than with a memory account.

Visualizing the perisaccadic shift of spatiotopic coordinates.

A point light source flickering on and off during a horizontal saccade projects a horizontal array onto the retina. The apparent visual direction of the tail end of the perceived (phantom) array reflects the amount of perisaccadic shift of spatiotopic coordinates that has been completed by the end of the saccade. Four men, saccading 8 degrees to the right across a flashing light, judged the horizontal visual direction of the left (tail) end of the phantom array relative to the left end of a standard 8 degrees array that had projected an image onto the retina before the saccade began. On average, the left ends appeared to be aligned when the last flash in the phantom array was imaged on the retina 7.4 degrees to the right of the image of the left end of the standard array. This result implies that the shift of spatiotopic coordinates is virtually complete by the end of the saccade.

Event-related brain potentials in a varied-set memory search task: a reconsideration.

Event-related potentials (ERPs) were recorded in 19 healthy subjects as they completed two Sternberg (1969, American Scientist, 57, 421-457) memory tests. In separate sessions, either single digits (i.e., 0-9) or 10 abstract figures were used as stimuli. In both sessions, memory set sizes were 1 (M1), 2 (M2), or 4 (M4). The amplitude and latency of the parietal P400 and the frontocentral negativity preceding P400 varied significantly with set size, but only between M1 and M2, whereas reaction time increased dramatically from M1 to M2 and from M2 to M4. These findings challenge previous assertions that the ERPs reflect aspects of the exhaustive serial search proposed by Sternberg. A late parietal positivity (P620), which failed to vary with set size, was larger in response to figures than to digits and may represent the search for, or utilization of, semantic traces of the stimuli.

Evoked brain potentials and memory: more positivity in response to forgotten items.

Brain evoked potentials (EPs) were recorded in human subjects participating in a free recall memory task involving retroactive interference. A learning list was presented five times. The fifth repetition was followed by an interference list. Both lists were composed of either words or abstract figures, and each subject experienced each of four possible combinations. Analysis of items recalled during the learning phase revealed larger N400 and P600 amplitudes for those items that were later forgotten vs remembered following the interference. This contradicts the usual finding that more positivity is associated with better memory. However, both the present as well as the extant findings can be explained in terms of cognitive resource allocation. Specifically, items receiving greater allocations are more likely to be immediately recalled. However, as the number of items in working memory increases, the allocation required to add new items also increases. Thus, items learned on later trials would receive larger allocations (i.e., larger positivities) than items learned earlier, yet would be more likely forgotten following the interference because their presence in memory would not be reinforced during later trials, as is the case with items learned earlier.

Timing the shift in retinal local signs that accompanies a saccadic eye movement.

The phantom array was used to probe the time course of the shift in retinal local signs that accompanies a saccadic eye movement. The phantom array materializes when one saccades in the dark across a point light source blinking 120 times per second. One sees a stationary array of flashes--the first materializes discretely near the intended endpoint of the saccade, and subsequent flashes materialize progressively closer to the actual position of the blinking light. Four trained observers indicated the perceived location, relative to the phantom array, of a 1-msec marker flash (M) produced by two LEDs (light-emitting diodes) that vertically bracketed the blinking light. The marker was seen as spatially coincident with the first flash when it flashed 80 to 0 msec before the saccade, and was seen as spatially coincident with either the first flash or the actual position of the blinking light when it flashed more than 80 msec before the saccade, indicating, respectively, that the shift is presaccadic and rather abrupt.

Pyriform sinus perforation secondary to traumatic intubation in a difficult airway patient.

Pyriform sinus perforation is a relatively rare complication of endotracheal intubation in anesthesia. It can occur after traumatic intubation and is potentially lethal. We recently encountered a patient who developed perforation of the left pyriform sinus when intubation was performed emergently by an inexperienced person in a difficult situation outside the operating room. Clinical manifestations and implications of this complication are discussed.

Minnesota Multiphasic Personality Inventory (MMPI) cluster groups among chronically ill patients: relationship to illness adjustment and treatment outcome.

Cluster analysis of the MMPI has been utilized widely in the chronic low back pain literature to try to identify reliable patient subtypes predictive of treatment outcome. We extended this methodology to patients with heterogeneous chronic medical conditions by replicating prototypic MMPI cluster group profiles and by relating cluster groups to clinical baseline and outcome data. Subjects were two independent samples (n = 254 and n = 263) of chronically ill patients admitted to an inpatient medicine/psychiatry unit. Using a four-cluster solution, similar cluster profile groups were replicated in both samples. Consistent differences emerged between cluster groups on functional impairment, psychiatric diagnoses, depression, and psychosomatic symptoms. Cluster group membership also predicted changes in functional impairment and depression six months after treatment. Results are discussed in terms of similarities between chronic low back pain and chronic illness and tailoring treatment to different patient types.

Depression and level of functioning in patients with rheumatoid arthritis.

This study examined the degree to which depression is related to physical and psychosocial dysfunction. The Beck Depression Inventory and the Sickness Impact Profile were administered to 34 patients with rheumatoid arthritis. Information on the demographic variables (age and employment status) and medical status variables (duration of disease and functional classification) were collected for each patient. Regression analyses revealed that depression was an important predictor of total, physical and psychosocial sickness-related behavioural dysfunction. The proportion of variance attributed to depression was moderate to large and was significant even after controlling for important demographic and medical status variables. These results suggest that depression is an important factor to be considered when evaluating the clinical significance of physical and psychosocial dysfunction in patients with rheumatoid arthritis.

The impact of psychologic factors on measurement of functional status. Assessment of the sickness impact profile.

In this study the relationship between four psychologic health constructs (depression, anxiety, patient response bias, and hostility) and the Sickness Impact Profile (SIP) measurement of functional status was evaluated. The SIP, Carroll Depression Rating Scale (CDRS); and the Minnesota Multiphasic Personality Inventory (MMPI) were administered to 332 patients hospitalized for treatment of combined medical and psychiatric problems. Pearson's product-moment correlation was high between CDRS and SIP Total score (r = 0.67) and between CDRS and SIP Psychosocial subscale (r = 0.72); correlation was lower between CDRS and SIP Physical subscale (r = 0.44). Six MMPI scales (depression, anxiety, psychasthenia, lie, K, hostility) correlated with SIP Total score (r = 0.18 to 0.50), with SIP Psychosocial score (r = 0.28 to 0.65) and less well with SIP Physical subscale (r = 0.07 to 0.25). Factor analysis of the SIP categories showed two factors with eigenvalues greater than 1. Promax factor rotation showed all SIP Psychosocial categories and all measured psychologic variables loaded most heavily on factor 1. SIP Physical categories loaded most heavily on factor 2. Stepwise multiple regression analysis showed that psychologic variables account for 49% of the SIP total variance, 62% of SIP Psychosocial subscale variance, but only 19% of SIP Physical subscale variance. The CDRS accounts for the major portion of the explained variance with only minor additional contributions from the MMPI scales. We conclude that 1) the SIP discriminates psychosocial and physical dysfunction even in medical patients with extensive psychiatric comorbidity; 2) the SIP measures at least two dimensions of health, one of which is strongly related to depression; and 3) constructs measured by MMPI scales do not have substantial independent contribution to SIP variance.

The relationship of appraisal and coping to chronic illness adjustment.

There is evidence that adaptation to chronic illness may be affected by psychological factors, especially how patients appraise and cope with the stress of their illness. The purpose of the present study was to examine the relationship of stress appraisal and coping responses to multiple behavioral indices of illness adjustment among patients with diverse chronic medical conditions. One hundred and one patients admitted to a multidisciplinary medicine/psychiatry unit completed measures of functional impairment, depression, symptom severity, and the Ways of Coping Checklist--Revised. Hierarchical regression analyses indicated that emotion-focused coping was positively related to poor psychosocial adjustment and depression after controlling for physician rated disease severity. Appraising chronic illness as holding one back predicted greater emotion-focused coping responses and poorer adjustment to illness. The use of problem-focused coping strategies was generally unrelated to illness adjustment. These findings suggest the presence of an emotion-focused coping triad consisting of wishful thinking, self blame, and avoidance, all of which appear to be maladaptive strategies when coping with chronic medical conditions. Implications for coping skills training and the need for longitudinal research is discussed.

Myelitis in a cat infected with Toxoplasma gondii and feline immunodeficiency virus.

Severe necrotizing myelitis secondary to localization and reactivation of Toxoplasma gondii within the spinal cord of a domestic shorthair cat was diagnosed by use of light and electron microscopy and immunohistochemistry. The cat also was infected with feline immunodeficiency virus. This case may have useful comparative features to T gondii infections in human patients with acquired immunodeficiency syndrome.