Highlighting Unseen Activity Through 48-Hour Continuous Measurement in Subacute Stroke Rehabilitation: Preliminary Cohort Study.

BACKGROUND: Motor impairments not only lead to a significant reduction in patient activity levels but also trigger a further deterioration in motor function due to deconditioning, which is an issue that is particularly pronounced during hospitalization. This deconditioning can be countered by sustaining appropriate activity levels. Activities that occur outside of scheduled programs, often overlooked, are critical in this context. Wearable technology, such as smart clothing, provides a means to monitor these activities. OBJECTIVE: This study aimed to observe activity levels in patients who had strokes during the subacute phase, focusing on both scheduled training sessions and other nontraining times in an inpatient rehabilitation environment. A smart clothing system is used to simultaneously measure heart rate and acceleration, offering insights into both the amount and intensity of the physical activity. METHODS: In this preliminary cohort study, 11 individuals undergoing subacute stroke rehabilitation were enrolled. The 48-hour continuous measurement system, deployed at admission and reassessed 4 weeks later, monitored accelerometry data for physical activity (quantified with a moving SD of acceleration [MSDA]) and heart rate for intensity (quantified with percent heart rate reserve). The measurements were performed using a wearable activity monitoring system, the hitoe (NTT Corporation and Toray Industries, Inc) system comprising a measuring garment (wear or strap) with integrated electrodes, a data transmitter, and a smartphone. The Functional Independence Measure was used to assess the patients' daily activity levels. This study explored factors such as differences in activity during training and nontraining periods, correlations with activities of daily living (ADLs) and age, and changes observed after 4 weeks. RESULTS: A significant increase was found in the daily total MSDA after the 4-week program, with the average percent heart rate reserve remaining consistent. Physical activity during training positively correlated with ADL levels both at admission (ρ=0.86, P<.001) and 4 weeks post admission (ρ=0.96, P<.001), whereas the correlation between age and MSDA was not significant during training periods at admission (ρ=-0.41, P=.21) or 4 weeks post admission (ρ=-0.25, P=.45). Conversely, nontraining activity showed a negative correlation with age, with significant negative correlations with age at admission (ρ=-0.82, P=.002) and 4 weeks post admission (ρ=-0.73, P=.01). CONCLUSIONS: Inpatient rehabilitation activity levels were positively correlated with ADL levels. Further analysis revealed a strong positive correlation between scheduled training activities and ADL levels, whereas nontraining activities showed no such correlation. Instead, a negative correlation between nontraining activities and age was observed. These observations suggest the importance of providing activity opportunities for older patients, while it may also suggest the need for adjusting the activity amount to accommodate the potentially limited fitness levels of this demographic. Future studies with larger patient groups are warranted to validate and further elucidate these findings.

Immediate Reduction in Spasticity of Ankle Plantar Flexors in a Stroke Patient after Treatment with a Spinning Permanent Magnet Device.

Background: Magnetic stimulation devices can be large because of the need for cooling systems. We developed a compact and lightweight Spinning Permanent Magnet (SPM) device that generates magnetic fields with intensities below the motor threshold. In this report, we present the case of a post-stroke patient in which an immediate reduction in spasticity of the ankle plantar flexors was achieved after SPM treatment. Case: A 37-year-old man was admitted to our hospital with a right putamen hemorrhage. The patient underwent conservative therapy and exhibited residual left hemiplegia and spasticity. Three months after stroke onset, he was able to walk with supervision while using a left ankle-foot orthosis and a T-cane. The Modified Ashworth Scale (MAS) score of the left ankle plantar flexors was 1+. The plantar flexors were stimulated by SPM treatment. The outcomes were the Hmax/Mmax of the tibial nerve (soleus muscle) and the MAS score. On the first day, SPM stimulation was applied for 30 min. On the second day, a sham stimulation of the same duration was performed. On the third day, the SPM stimulation was repeated. Hmax/Mmax decreased from 41.5% to 37.7% on the first day, and from 46.9% to 31.6% on the third day after SPM stimulation. The MAS score decreased from 1+ to 1 on both days. In contrast, after sham stimulation, Hmax/Mmax increased from 39.2% to 44.2%, whereas the MAS score remained unchanged at 1+. Discussion: Stimulation below the motor threshold using SPM treatment can effectively reduce spasticity.

Dynamic regulation of ubiquitylation and deubiquitylation at the central spindle during cytokinesis.

During cytokinesis, the central spindle, a bundle of interdigitated anti-parallel microtubules between separating chromosomes, recruits various cytokinetic regulator proteins to the cleavage region. Here, we show that the level of protein ubiquitylation is strikingly and transiently elevated in Aurora B kinase-positive double-band regions of the central spindle during cytokinesis. Two deubiquitylating enzymes UBPY and AMSH, which act on endosomes in interphase, were also recruited to the cleavage region. Whereas UBPY was detected only in the final stage of cytokinesis at the midbody, AMSH localized to a ring structure surrounding the mitotic kinesin MKLP1-positive region of the central spindle and midbody throughout cytokinesis. Depletion of cellular UBPY or AMSH led to defects in cytokinesis. VAMP8, a v-SNARE required for vesicle fusion in cytokinesis, localized to the central spindle region positive for ubiquitylated proteins, and underwent ubiquitylation and deubiquitylation by both UBPY and AMSH. Our results thus implicate the ubiquitylation/deubiquitylation of proteins including VAMP8 in cytokinesis.

14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase.

The deubiquitinating enzyme UBPY, also known as USP8, regulates cargo sorting and membrane traffic at early endosomes. Here we demonstrate the regulatory mechanism of the UBPY catalytic activity. We identified 14-3-3 epsilon, gamma, and zeta as UBPY-binding proteins using co-immunoprecipitation followed by mass spectrometric analysis. The 14-3-3 binding of UBPY was inhibited by mutating the consensus 14-3-3-binding motif RSYS(680)SP, by phosphatase treatment, and by competition with the Ser(680)-phosphorylated RSYS(680)SP peptide. Metabolic labeling with [(32)P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser(680) is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser(680) in a phosphorylation-dependent manner. The mutation at Ser(680) led to enhanced ubiquitin isopeptidase activity of UBPY toward poly-ubiquitin chains and a cellular substrate, epidermal growth factor receptor, in vitro and in vivo. Moreover, addition of 14-3-3epsilon inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser(680) and dissociated from 14-3-3s in the M phase, resulting in enhanced activity of UBPY during cell division. We conclude that UBPY is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3s during the interphase, and this regulation is cancelled in the M phase.

A deubiquitinating enzyme UBPY regulates the level of protein ubiquitination on endosomes.

Monoubiquitination of endocytosed cell surface receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. The sorting of ubiquitinated proteins is executed by concerted actions of class E vacuolar protein sorting (Vps) proteins. Some proteins in the sorting machinery undergo monoubiquitination, suggesting that their functions are also regulated by ubiquitination. The Hrs-STAM complex, a class E Vps protein complex essential for the initial step of the sorting pathway, binds two deubiquitinating enzymes, UBPY and AMSH. Here we examined the effects of inactivating UBPY on protein ubiquitination at endosomes. Overexpression of a catalytically inactive UBPY mutant or depletion of UBPY by RNA interference resulted in the accumulation of ubiquitinated proteins on morphologically aberrant endosomes. Electron microscopy showed that they are aggregates of multivesicular endosomes. Among the sorting machinery proteins that undergo ubiquitination, Eps15 was monoubiquitinated at an elevated level in UBPY-inactivated cells. UBPY also deubiquitinated Eps15 in vitro, suggesting that Eps15 is a cellular substrate for UBPY. Furthermore, inactivation of UBPY caused the accumulation of Eps15 on the endosomal aggregates. These results suggest that UBPY regulates the level of protein ubiquitination on endosomes, which is required for maintaining the morphology of the organelle.

Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes.

Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This "receptor down-regulation" process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.

A role for Hrs in endosomal sorting of ligand-stimulated and unstimulated epidermal growth factor receptor.

Ligand-stimulated growth factor receptors are rapidly internalized and transported to early endosomes. Unstimulated receptors are also internalized constitutively, although at a slower rate, and delivered to the same organelle. At early endosomes, stimulated receptors are sorted for the lysosomal degradation pathway, whereas unstimulated receptors are mostly recycled back to the cell surface. To investigate the role of Hrs, an early endosomal protein, in this sorting process, we overexpressed Hrs in HeLa cells and examined the intracellular trafficking of epidermal growth factor receptor (EGFR) in EGF-stimulated and unstimulated cells. Overexpression of Hrs inhibited the trafficking of EGFR from early endosomes, resulting in an accumulation of EGFR on early endosomes in both ligand-stimulated and unstimulated cells. On the other hand, overexpression of Hrs mutants with a deletion or a point mutation within the FYVE domain did not inhibit the trafficking. These results suggest that Hrs regulates the sorting of ligand-stimulated and unstimulated growth factor receptors on early endosomes, and that the FYVE domain, which is required for Hrs to reside in a microdomain of early endosomes, plays an essential role in the function of Hrs.

Association with Hrs is required for the early endosomal localization, stability, and function of STAM.

Members of the STAM family of proteins, STAM1 and STAM2, are associated with Hrs through their coiled-coil regions. Both Hrs and STAM bind ubiquitin and are involved in endosomal sorting of ubiquitinated cargo proteins for trafficking to the lysosome. Here we examined the biological significance of STAM binding to Hrs. Endogenous STAM1 and STAM2 were mostly localized on the early endosome, suggesting that they are resident endosomal proteins. A STAM2 mutant that lacks the coiled-coil region and does not bind Hrs, in contrast, mislocalized to the cytoplasm. Deletion of a region located N-terminal to the coiled-coil region and conserved among STAM proteins also severely affected Hrs binding and the endosomal localization of STAM2, suggesting that this region is also involved in these activities. Depletion of endogenous Hrs by RNA interference similarly caused the mislocalization of exogenously expressed STAM2 to the cytoplasm. These results indicate that STAM is localized to the early endosome by binding to Hrs on the target membrane. In addition, the expression level of endogenous STAM proteins was drastically reduced in Hrs-depleted cells, suggesting that STAM is stabilized by binding to Hrs. Finally, STAM2 mutants lacking the Hrs-binding activity were defective in causing the enlargement of early endosomes, accumulating ubiquitinated proteins on this aberrant organelle, and inhibiting the degradation of ligand-activated epidermal growth factor receptors, suggesting that the association with Hrs is a prerequisite for STAM function.

STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif.

Conjugation with ubiquitin acts as a sorting signal for proteins in the endocytic and biosynthetic pathways at the endosome. Signal-transducing adaptor molecule (STAM) proteins, STAM1 and STAM2, are associated with hepatocyte growth factor-regulated substrate (Hrs) but their function remains unknown. Herein, we show that STAM proteins bind ubiquitin and ubiquitinated proteins and that the tandemly located VHS (Vps27/Hrs/STAM) domain and ubiquitin-interacting motif serve as the binding site(s). STAM proteins colocalize with Hrs on the early endosome. Overexpression of STAM proteins, but not their mutants lacking the ubiquitin-binding activity, causes the accumulation of ubiquitinated proteins and ligand-activated epidermal growth factor receptor on the early endosome. These results suggest that through interaction with ubiquitinated cargo proteins on the early endosome via the VHS domain and ubiquitin-interacting motif, STAM proteins participate in the sorting of cargo proteins for trafficking to the lysosome.