Temporal Pattern of Cortical Hypoxia in Multiple Sclerosis and Its Significance on Neuropsychological and Clinical Measures of Disability.

OBJECTIVE: Multiple sclerosis (MS) is a degenerative disease of the central nervous system (CNS) characterized by inflammation, demyelination, and axonal damage. It has been hypothesized that hypoxia plays a role in the pathogenesis of MS. This study was undertaken to investigate the reproducibility of non-invasively measured cortical microvascular hemoglobin oxygenation (St O2 ) using frequency domain near-infrared spectroscopy (fdNIRS), investigate its temporal pattern of hypoxia in people with MS (pwMS), and its relationship with neurocognitive function and mood. METHODS: We investigated the reproducibility of fdNIRS measurements. We measured cortical hypoxia in pwMS, and the relationships between St O2 , neurocognitive function, fatigue, and measures of physical disability. Furthermore, we cataloged the temporal pattern of St O2 measured at 1-week intervals for 4 weeks, and at 8 weeks and ~1 year. RESULTS: We show that fdNIRS parameters were highly reproducible in 7 healthy control participants measured over 6 days (p > 0.05). There was low variability between and within subjects. In line with our previous findings, we show that 33% of pwMS (n = 88) have cortical microvascular hypoxia. Over 8 weeks and at ~1 year, St O2 values for normoxic and hypoxic groups did not change significantly. There was no significant association between cognitive function and St O2 . This conclusion should be revisited as only a small proportion of the relapsing-remitting MS group (21%) was cognitively impaired. INTERPRETATION: The fdNIRS parameters have high reproducibility and repeatability, and we have demonstrated that hypoxia in MS is a chronic condition, lasting at least a year. The results show a weak relationship between cognitive functioning and oxygenation, indicating future study is required. ANN NEUROL 2023;94:1067-1079.

Discovery of Benzodiazepine-Based Inhibitors of the E2 Enzyme UBCH10 from a Cell-Based p21 Degradation Screen.

p21Cip1 (p21) is a universal cyclin-dependent kinase (CDK) inhibitor that halts cell proliferation and tumor growth by multiple mechanisms. The expression of p21 is often downregulated in cancer cells as a result of the loss of function of transcriptional activators, such as p53, or the increased degradation rate of the protein. To identify small molecules that block the ubiquitin-mediated degradation of p21 as a future avenue for cancer drug discovery, we have screened a compound library using a cell-based reporter assay of p21 degradation. This led to the identification of a benzodiazepine series of molecules that induce the accumulation of p21 in cells. Using a chemical proteomic strategy, we identified the ubiquitin-conjugating enzyme UBCH10 as a cellular target of this benzodiazepine series. We show that an optimized benzodiazepine analogue inhibits UBCH10 ubiquitin-conjugating activity and substrate proteolysis by the anaphase-promoting complex.

NIRS Studies Show Reduced Interhemispheric Functional Connectivity in Individuals with Multiple Sclerosis That Exhibit Cortical Hypoxia.

Many with multiple sclerosis (MS) have low cortical microvascular oxygen levels (hypoxia), which have been previously proposed to exacerbate inflammation in MS. We do not know if hypoxia impacts or relates to brain function. We hypothesise that within the MS population, those who have hypoxia may show reduced brain functional connectivity (FC). We recruited 20 MS participants and grouped them into normoxic and hypoxic groups (n = 10 in each group) using frequency-domain near-infrared spectroscopy (fdNIRS). Functional coherence of the haemodynamic signal, quantified with functional near-infrared spectroscopy (fNIRS) was used as a marker of brain function and was carried out during resting-state, finger-tapping, and while completing two neurocognitive tasks. Reduced FC was detected in the hypoxic MS group. fNIRS measures of haemodynamic coherence in MS could be a biomarker of functional impairment and/or disease progression.

Multiple sclerosis-related fatigue: the role of impaired corticospinal responses and heightened exercise fatigability.

It is unclear whether motor fatigability and perceived fatigue share a common pathophysiology in people with multiple sclerosis (PwMS). This cross-sectional investigation explored the relationship between the mechanisms of motor fatigability from cycling and fatigue severity in PwMS. Thirteen highly fatigued (HF) and thirteen nonfatigued (LF) PwMS and thirteen healthy controls (CON) completed a step test until volitional exhaustion on an innovative cycle ergometer. Neuromuscular evaluations involving femoral nerve electrical stimulation and transcranial magnetic stimulation were performed every 3 min throughout cycling. One-way ANOVA at baseline and exhaustion uncovered evidence of consistently smaller motor evoked potential (MEP) amplitudes (P = 0.011) and prolonged MEP latencies (P = 0.041) in HF as well as a greater decline in maximal voluntary contraction force (HF: 63 ± 13%; LF: 75 ± 13%; CON: 73 ± 11% of pre; P = 0.037) and potentiated twitch force (HF: 35 ± 13%; LF: 50 ± 16%; CON: 47 ± 17% of pre; P = 0.049) in HF at volitional exhaustion. Hierarchical regression determined that fatigue severity on the Fatigue Severity Scale was predicted by prolonged MEP latencies (change in r2 = 0.389), elevated peripheral muscle fatigability (change in r2 = 0.183), and depressive symptoms (change in r2 = 0.213). These findings indicate that MS-related fatigue is distinguished by disrupted corticospinal responsiveness, which could suggest progressive pathology, but fatigability from whole body exercise and depressive symptoms also influence perceptions of fatigue in PwMS.NEW & NOTEWORTHY The etiology of fatigability from whole body exercise was examined for the first time to accurately elucidate the relationship between fatigue and fatigability in multiple sclerosis (MS). Compromised corticospinal responsiveness predicted fatigue severity, providing a novel, objective indicator of fatigue in MS. Although the impaired corticomotor transmission did not aggravate muscle activation in this group of people with multiple sclerosis (PwMS) of lower disability, heightened muscle fatigability was seen to contribute to perceptions of fatigue in PwMS.

Reliability and validity of a novel device for quantifying ankle dorsiflexion force in persons with multiple sclerosis.

BACKGROUND: With emerging treatment modalities and therapeutics for Multiple Sclerosis (MS), there is a critical need for improved measures of disability. Routine clinical practice and trials will benefit from devices that are capable of objectively quantifying muscle strength/weakness. We have developed a device for measuring Tibialis Anterior (TA) force that is both objective and easy to use - the Rapid Objective Quantification - TA (ROQ-TA). The purpose of this study was to determine the reliability and validity of the ROQ-TA versus Manual Muscle Testing and Isokinetic Dynamometry (IKD) for evaluating TA force in persons with MS (PwMS). METHODS: Ankle dorsiflexion of 20 PwMS was assessed by three modalities: ROQ-TA, MMT, and IKD over 2 testing sessions. ICC(2,1) values and Bland-Altman plots were used to assess reliability and validity of the ROQ-TA. RESULTS: The ICC(2,1) for reliability for the ROQ-TA was found to be 0.884 (0.690-0.957) while the IKD produced a similar ICC(2,1) of 0.919 (0.784-0.970). The mean difference between the two sessions for the ROQ-TA was -6.4 N with limits of agreement of 42.5 to -55.4 N as inferred by the Bland-Altman plots. With respect to validity, the ROQ-TA versus IKD yielded similar values for both sessions- the mean bias was 9.3 N (SE range: -3.4 to 22 N) for session 1 and 9.9 N for session 2 (SE range: -3.2 to 23.0 N). The ICC(2,1) values between the two devices were in moderate agreement - session 1: 0.579 (-0.125-0.843) and session 2: 0.490 (-0.363-0.809). CONCLUSION: The ROQ-TA is a valid and highly reliable device to test dorsiflexion force in PwMS.

Portable fixed dynamometry to quantify ankle dorsiflexion force.

INTRODUCTION: Quantifying muscle strength is critical in clinical and research settings. A rapid and objective method is ideal. The primary objective of this study was to examine the reliability of a novel device, the rapid objective quantification- tibialis anterior (ROQ-TA), which quantifies the dorsiflexion force of the tibialis anterior, and to assess its validity against isokinetic dynamometry (IKD). METHODS: Ankle dorsiflexion of 20 healthy subjects was assessed by 3 modalities, ROQ-TA, manual muscle testing, and isokinetic dynamometry, over 2 testing sessions. RESULTS: The intraclass correlation coefficient [ICC(2,1) ] for reliability was 0.872 (0.677-0.949) for the ROQ-TA and 0.892 (0.728-0.957) for IKD. For validity, the ICC(2,1) values for the ROQ-TA and IKD were in good agreement, with 0.672 (0.17-0.87) in the first testing session and 0.769 (0.42-0.91) in the second session. DISCUSSION: The ROQ-TA is a valid and reliable device to test ankle dorsiflexion force in a healthy population. Muscle Nerve, 2018.

Long-term persistence with injectable therapy in relapsing-remitting multiple sclerosis: an 18-year observational cohort study.

Disease modifying therapies (DMTs) reduce the frequency of relapses and accumulation of disability in multiple sclerosis (MS). Long-term persistence with treatment is important to optimize treatment benefit. This long-term, cohort study was conducted at the Calgary MS Clinic. All consenting adults with relapsing-remitting MS who started either glatiramer acetate (GA) or interferon-ß 1a/1b (IFN-ß) between January 1st, 1996 and July 1st, 2011 were included. Follow-up continued to February 1st, 2014. Time-to-discontinuation of the initial and subsequently-prescribed DMTs (switches) was analysed using Kaplan-Meier survival analyses. Group differences were compared using log-rank tests and multivariable Cox regression models. Analysis included 1471 participants; 906 were initially prescribed GA and 565 were initially prescribed IFN-ß. Follow-up information was available for 87%; 29 (2%) were lost to follow-up and 160 (11%) moved from Southern Alberta while still using DMT. Median time-to-discontinuation of all injectable DMTs was 11.1 years. Participants with greater disability at treatment initiation, those who started treatment before age 30, and those who started between 2006 and 2011 were more likely to discontinue use of all injectable DMTs. Median time-to-discontinuation of the initial DMT was 8.6 years. Those initially prescribed GA remained on treatment longer. Of 610 participants who discontinued injectable DMT, 331 (54%) started an oral DMT, or a second-line DMT, or resumed injectable DMT after 90 days. Persistence with injectable DMTs was high in this long-term population-based study. Most participants who discontinued injectable DMT did not remain untreated. Further research is required to understand treatment outcomes and outcomes after stopping DMT.

AKAP79 modulation of L-type channels involves disruption of intramolecular interactions in the CaV1.2 subunit.

L-type voltage gated calcium channels (VGCCs) interact with a variety of proteins that modulate both their function and localization. A-Kinase Anchoring Proteins (AKAPs) facilitate L-type calcium channel phosphorylation through ß adrenergic stimulation. Our previous work indicated a role of neuronal AKAP79/150 in the membrane targeting of Ca(V)1.2 L-type calcium channels, which involved a proline rich domain (PRD) in the intracellular II-III loop of the channel.(1) Here, we show that mutation of proline 857 to alanine (P857A) into the PRD does not disrupt the AKAP79-induced increase in Ca(v)1.2 membrane expression. Furthermore, deletion of two other PRDs into the carboxy terminal domain of Ca(V)1.2 did not alter the targeting role of AKAP79. In contrast, the distal carboxy terminus region of the channel directly interacts with AKAP79. This protein-protein interaction competes with a direct association of the channel II-III linker on the carboxy terminal tail and modulates membrane targeting of Ca(V)1.2. Thus, our results suggest that the effects of AKAP79 occur through relief of an autoinhibitory mechanism mediated by intramolecular interactions of Ca(v)1.2 intracellular regions.

Bipartite syntaxin 1A interactions mediate CaV2.2 calcium channel regulation.

Functional interactions between syntaxin 1A and Ca(V)2 calcium channels are critical for fast neurotransmitter release in the mammalian brain, and coexpression of syntaxin 1A with these channels not only regulates channel availability, but also promotes G-protein inhibition. Both the syntaxin 1A C-terminal H3 domain, and N-terminal Ha domain have been shown to interact with the Ca(V)2.2 channel synprint region, suggesting a bipartite model of functional interaction, however the molecular determinants of this interaction have not been closely investigated. We used in vitro binding assays to assess interactions of syntaxin 1A truncation mutants with Ca(V)2.2 synprint and Ca(V)2.3 II-III linker regions. We identified two distinct interactions between the Ca(V)2.2 synprint region and syntaxin 1A: the first between C-terminal H3c domain of syntaxin 1A and residues 822-872 of Ca(V)2.2; and the second between the N-terminal 10 residues of the syntaxin 1A Ha region and residues 718-771 of Ca(V)2.2. The N-terminal syntaxin 1A fragment also interacted with the Ca(V)2.3 II-III linker. We then performed whole cell patch clamp recordings to test the effects of a putative interacting syntaxin 1A N-terminus peptide with Ca(V)2.2 and Ca(V)2.3 channels in a recombinant expression system. A YFP-tagged peptide corresponding to the N-terminal 10 residues of the syntaxin 1A Ha domain was sufficient to allosterically inhibit both Ca(V)2.2 and Ca(V)2.3 channel function but had no effect on G-protein mediated inhibition. Our results support a model of bipartite functional interactions between syntaxin 1A and Ca(V)2.2 channels and add accuracy to the two putative interacting domains, consistent with previous studies. Furthermore, we highlight the syntaxin 1A N-terminus as the minimal determinant for functional regulation of Ca(V)2.2 and Ca(V)2.3 channels.

Synthesis of enantiopure substituted piperidines via an aziridinium ring expansion.

Herein we report a novel methodology for the asymmetric synthesis of 3-substituted piperidines from readily available chiral building blocks. This method, which features a novel irreversible dihydropyrole-tetrahydropyridine ring expansion, allows the introduction of a large variety of substituents at the 3-position and permits substitution at the 2- and 6-position giving mono-, di-, or trisubstituted piperidines with high diastereocontrol.

MTLD, vocd-D, and HD-D: a validation study of sophisticated approaches to lexical diversity assessment.

The main purpose of this study was to examine the validity of the approach to lexical diversity assessment known as the measure of textual lexical diversity (MTLD). The index for this approach is calculated as the mean length of word strings that maintain a criterion level of lexical variation. To validate the MTLD approach, we compared it against the performances of the primary competing indices in the field, which include vocd-D, TTR, Maas, Yule's K, and an HD-D index derived directly from the hypergeometric distribution function. The comparisons involved assessments of convergent validity, divergent validity, internal validity, and incremental validity. The results of our assessments of these indices across two separate corpora suggest three major findings. First, MTLD performs well with respect to all four types of validity and is, in fact, the only index not found to vary as a function of text length. Second, HD-D is a viable alternative to the vocd-D standard. And third, three of the indices--MTLD, vocd-D (or HD-D), and Maas--appear to capture unique lexical information. We conclude by advising researchers to consider using MTLD, vocd-D (or HD-D), and Maas in their studies, rather than any single index, noting that lexical diversity can be assessed in many ways and each approach may be informative as to the construct under investigation.

The perceptions and habits of alcohol consumption and smoking among Canadian medical students.

OBJECTIVE: The authors aim to quantify the extent, and to assess student perception, of alcohol and tobacco use among medical students at the University of Calgary, and the relationship of these attitudes to problem drinking (according to the CAGE questionnaire). METHODS: A questionnaire was distributed to first-, second-, and third-year medical students attending the University of Calgary medical school. RESULTS: Of the 327 students enrolled, 175 of students responded to the questionnaire. Six percent of the students currently smoke while 24% of students reported cigarette smoking at some point in their life. Eighty-six percent of students currently drink, with a majority drinking fewer than 11 drinks per week. Fifteen percent of students were at an increased risk for problem drinking according to the CAGE questionnaire. An increased risk for problem drinking was significantly related to believing more strongly that getting drunk is acceptable on occasion and less strongly that increased alcohol has many negative health consequences, as well as feeling less in control of alcohol consumption. CONCLUSION: Medical students at the University of Calgary consume less alcohol and cigarettes than a comparable population. However, a high proportion of students are at risk for alcohol abuse according to the CAGE questionnaire. The results of this study suggest that although the quantity of alcohol consumed is not a substantial concern at this time, students might be at risk for future alcohol abuse.

Trafficking and regulation of neuronal voltage-gated calcium channels.

The importance of voltage-gated calcium channels is underscored by the multitude of intracellular processes that depend on calcium, notably gene regulation and neurotransmission. Given their pivotal roles in calcium (and hence, cellular) homeostasis, voltage-gated calcium channels have been the subject of intense research, much of which has focused on channel regulation. While ongoing research continues to delineate the myriad of interactions that govern calcium channel regulation, an increasing amount of work has focused on the trafficking of voltage-gated calcium channels. This includes the mechanisms by which calcium channels are targeted to the plasma membrane, and, more specifically, to their appropriate loci within a given cell. In addition, we are beginning to gain some insights into the mechanisms by which calcium channels can be removed from the plasma membrane for recycling and/or degradation. Here we highlight recent advances in our understanding of these fundamentally important mechanisms.

Masters or slaves? Vesicle release machinery and the regulation of presynaptic calcium channels.

Calcium entry through presynaptic voltage-gated calcium channels is essential for neurotransmitter release. The two major types of presynaptic calcium channels contain a synaptic protein interaction site that physically interacts with synaptic vesicle release proteins. This is thought to tighten the coupling between the sources of calcium entry and the neurotransmitter release machinery. Conversely, the binding of synaptic proteins to presynaptic calcium channels regulates calcium channel activity. Hence, presynaptic calcium channels act not only as the masters of the synaptic release process, but also as key targets for feedback inhibition.

Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit.

Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.

Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage-dependent inactivation of HVA calcium channels.

Voltage-dependent inactivation of calcium channels is a key mechanism for regulating intracellular calcium levels and neuronal excitability. In sodium and potassium channels, the molecular determinants that govern fast inactivation involve pore block by a cytoplasmic gating particle. As we discuss here, there is an increasing body of evidence that is consistent with a qualitatively similar inactivation mechanism in high-voltage-activated calcium channels. Work from a number of laboratories has implicated both cytoplasmic regions and the pore-lining S6 transmembrane helices in the inactivation process. Together with our recent findings, this leads us to propose a model in which the intracellular domain I-II linker region acts as a 'hinged lid' that physically occludes the pore by docking to the cytoplasmic ends of the S6 segments. We further propose that the ancillary calcium channel Beta subunits differentially modulate inactivation kinetics by binding to and thereby regulating the mobility of the putative inactivation gate. Indeed, additional evidence suggests that the carboxy terminus, amino terminus and domain III-IV linker regions of the channel modulate inactivation rates through interactions with the I-II linker per se, or indirectly via the ancillary Beta subunits. Taken together, the fast voltage-dependent inactivation of calcium channels appears reminiscent of that of sodium channels, but appears to show a more complex regulation through intramolecular interactions between the putative inactivation gate and other cytoplasmic regions.

Molecular determinants of cysteine string protein modulation of N-type calcium channels.

Cysteine string proteins (CSPs) are secretory vesicle chaperones that are important for neurotransmitter release. We have previously reported an interaction of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels that results in a tonic G protein inhibition of the channels. In this report we directly demonstrate that two separate regions of CSP associate with G proteins. The N-terminal binding site of CSP, which includes the J domain, binds Galpha subunits but not Galphabeta subunits whereas the C terminal binding site of CSP associates with either free Galphabeta subunits or Galphabeta in complex with Galpha. The interaction of either binding site of CSP (CSP1-82 or CSP83-198) with G proteins elicits robust tonic inhibition of N-type calcium channel activity. However, CSP1-82 inhibition and CSP83-198 inhibition of calcium channels occur through distinct mechanisms. Calcium channel inhibition by CSP83-198 (but not CSP1-82) is completely blocked by co-expression of the synaptic protein interaction site (synprint) of the N-type channel, indicating that CSP83-198 inhibition is dependent on a physical interaction with the calcium channel. These results suggest that distinct binding sites of CSP can play a role in modulating G protein function and G protein inhibition of calcium channels.

Calcium-triggered membrane fusion proceeds independently of specific presynaptic proteins.

Complexes of specific presynaptic proteins have been hypothesized to drive or catalyze the membrane fusion steps of exocytosis. Here we use a stage-specific preparation to test the roles of SNAREs, synaptotagmin, and SNARE-binding proteins in the mechanism of Ca2+-triggered membrane fusion. Excess exogenous proteins, sufficient to block SNARE interactions, did not inhibit either the Ca2+ sensitivity, extent, or kinetics of fusion. In contrast, despite a limited effect on SNARE and synaptotagmin densities, treatments with high doses of chymotrypsin markedly inhibited fusion. Conversely, low doses of chymotrypsin had no effect on the Ca2+ sensitivity or extent of fusion but did alter the kinetic profile, indicating a more direct involvement of other proteins in the triggered fusion pathway. SNAREs, synaptotagmin, and their immediate binding partners are critical to exocytosis at a stage other than membrane fusion, although they may still influence the triggered steps.