Sungyoung Auh

Effective transvascular delivery of nanoparticles across the blood-brain tumor barrier into malignant glioma cells

BackgroundEffective transvascular delivery of nanoparticle-based chemotherapeutics across the blood-brain tumor barrier of malignant gliomas remains a challenge. This is due to our limited understanding of nanoparticle properties in relation to the physiologic size of pores within the blood-brain tumor barrier. Polyamidoamine dendrimers are particularly small multigenerational nanoparticles with uniform sizes within each generation. Dendrimer sizes increase by only 1 to 2 nm with each successive generation. Using functionalized polyamidoamine dendrimer generations 1 through 8, we investigated how nanoparticle size influences particle accumulation within malignant glioma cells.MethodsMagnetic resonance and fluorescence imaging probes were conjugated to the dendrimer terminal amines. Functionalized dendrimers were administered intravenously to rodents with orthotopically grown malignant gliomas. Transvascular transport and accumulation of the nanoparticles in brain tumor tissue was measured in vivo with dynamic contrast-enhanced magnetic resonance imaging. Localization of the nanoparticles within glioma cells was confirmed ex vivo with fluorescence imaging.ResultsWe found that the intravenously administered functionalized dendrimers less than approximately 11.7 to 11.9 nm in diameter were able to traverse pores of the blood-brain tumor barrier of RG-2 malignant gliomas, while larger ones could not. Of the permeable functionalized dendrimer generations, those that possessed long blood half-lives could accumulate within glioma cells.ConclusionThe therapeutically relevant upper limit of blood-brain tumor barrier pore size is approximately 11.7 to 11.9 nm. Therefore, effective transvascular drug delivery into malignant glioma cells can be accomplished by using nanoparticles that are smaller than 11.7 to 11.9 nm in diameter and possess long blood half-lives.

Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells

Hibernation torpor provides an excellent natural model of tolerance to profound reductions in blood flow to the brain and other organs. Here, we report that during torpor of 13-lined ground squirrels, massive SUMOylation occurs in the brain, liver, and kidney. The level of small ubiquitin-related modifier (SUMO) conjugation coincides with the expression level of Ubc9, the SUMO specific E2-conjugating enzyme. Hypothermia alone also increased SUMO conjugation, but not as markedly as hibernation torpor. Increased SUMO conjugation (induced by Ubc9 overexpression, ischemic preconditioning (PC) ± hypothermia) was necessary and sufficient for tolerance of SHSY5Y neuroblastoma cells to oxygen/glucose deprivation (OGD) (‘in vitro ischemia‘); decreased SUMO conjugation (induced by a dominant-negative Ubc9) severely reduced tolerance to OGD in these cells. These data indicate that post-translational modification of proteins by SUMOylation is a prominent feature of hibernation torpor and is critical for cytoprotection by ischemic PC ± hypothermia in SHSY5Y cells subjected to OGD.

Clinical features of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy is an X-linked motor neuron disease caused by a CAG repeat expansion in the androgen receptor gene. To characterize the natural history and define outcome measures for clinical trials, we assessed the clinical history, laboratory findings and muscle strength and function in 57 patients with genetically confirmed disease. We also administered self-assessment questionnaires for activities of daily living, quality of life and erectile function. We found an average delay of over 5 years from onset of weakness to diagnosis. Muscle strength and function correlated directly with serum testosterone levels and inversely with CAG repeat length, age and duration of weakness. Motor unit number estimation was decreased by about half compared to healthy controls. Sensory nerve action potentials were reduced in nearly all subjects. Quantitative muscle assessment and timed 2 min walk may be useful as meaningful indicators of disease status. The direct correlation of testosterone levels with muscle strength indicates that androgens may have a positive effect on muscle function in spinal and bulbar muscular atrophy patients, in addition to the toxic effects described in animal models.

Efficacy and safety of dutasteride in patients with spinal and bulbar muscular atrophy: a randomised placebo-controlled trial

BACKGROUND Spinal and bulbar muscular atrophy (SBMA) is caused by polyglutamine expansion in the androgen receptor, which results in ligand-dependent toxicity. Animal models have a neuromuscular deficit that is mitigated by androgen-reducing treatment. We aimed to assess the efficacy and safety of the 5α-reductase inhibitor dutasteride in patients with SBMA, and to identify outcome measures for use in future studies of the disease. METHODS We undertook a randomised, double-blind, placebo-controlled, single-site clinical trial in ambulatory, symptomatic men with genetically confirmed SBMA. Participants were assigned by random number table to receive dutasteride (0·5 mg per day) or placebo orally for 24 months. Patients and investigators were masked to treatment allocation. The primary outcome measure was quantitative muscle assessment (QMA). The final efficacy analysis included all patients who were compliant with study treatment at 24 months. This trial was registered with ClinicalTrials.gov, NCT00303446. FINDINGS 50 men were randomly assigned to treatment groups (25 dutasteride, 25 placebo), and 44 were included in the efficacy analysis (21 dutasteride, 23 placebo). At 24 months, the placebo group showed a decrease of 4·5% (-0·30 kg/kg) from baseline in weight-scaled muscle strength as indicated by QMA, and the dutasteride group had an increase in strength of 1·3% (0·14 kg/kg); the difference between groups (5·8%, 95% CI -5·9 to 17·6; p=0·28) was not significant. Prespecified secondary outcome measures of creatine kinase, muscle strength and function, motor nerve conduction, activities of daily living, and erectile function did not show a significant difference between the study groups in change from baseline. Quality of life, as measured by the physical component summary of the Medical Outcomes Study 36-item Short Form version 2, favoured dutasteride (change in score from baseline: placebo, -3·6%, vs dutasteride, 2·1%; p=0·01), whereas the mental component summary favoured placebo (3·3%vs -3·2%; p=0·03). The dutasteride group had fewer patients reporting falls than did the placebo group (9 vs 16; p=0·048); there were no other significant differences in reported adverse events. INTERPRETATION Our study did not show a significant effect of dutasteride on the progression of muscle weakness in SBMA, although there were secondary indications of both positive and negative effects compared with placebo. A longer trial duration or larger number of patients might be needed to show an effect on disease progression. Performance testing, QMA, and quality of life measures were identified as potentially useful endpoints for future therapeutic trials.

SUMOylation participates in induction of ischemic tolerance

Ground squirrels in hibernation torpor have been shown to have striking increases in global SUMOylation on tissue immunoblots. Here, we find evidence that global SUMOylation is also involved in ischemic tolerance in primary cortical neuronal cultures (from rats and mice) and SHSY5Y human neuroblastoma cells. Cultured cortical neurons preconditioned by sublethal oxygen/glucose deprivation (OGD) were less vulnerable to severe OGD than non‐preconditioned neurons. Preconditioned neurons maintained elevated SUMO‐1 conjugation levels (and, to a lesser extent those of SUMO‐2/3) on western blots in contrast to non‐preconditioned cells. Further, cortical neurons and SHSY5Y cells in which transfected SUMO‐1 or SUMO‐2 were over‐expressed showed increased survival after severe OGD. In contrast, cell cultures subjected to depletion of endogenous SUMO‐1 protein by RNAi had reduced survival after exposure to this form of in vitro ischemia and an attenuated protective response to preconditioning. These findings suggest that maintenance of a globally elevated SUMO‐1 (and maybe SUMO‐2/3) conjugation level as revealed by immunoblot assays is a component of ischemic tolerance.

Elevated global SUMOylation in Ubc9 transgenic mice protects their brains against focal cerebral ischemic damage.

We have previously shown that a massive increase in global SUMOylation occurs during torpor in ground squirrels, and that overexpression of Ubc9 and/or SUMO-1 in cell lines and cortical neurons protects against oxygen and glucose deprivation. To examine whether increased global SUMOylation protects against ischemic brain damage, we have generated transgenic mice in which Ubc9 is expressed strongly in all tissues under the chicken β-actin promoter. Ubc9 expression levels in 10 founder lines ranged from 2 to 30 times the endogenous level, and lines that expressed Ubc9 at modestly increased levels showed robust resistance to brain ischemia compared to wild type mice. The infarction size was inversely correlated with the Ubc9 expression levels for up to five times the endogenous level. Although further increases showed no additional benefit, the Ubc9 expression level was highly correlated with global SUMO-1 conjugation levels (and SUMO-2,3 levels to a lesser extent) up to a five-fold Ubc9 increase. Most importantly, there were striking reciprocal relationships between SUMO-1 (and SUMO-2,3) conjugation levels and cerebral infarction volumes among all tested animals, suggesting that the limit in cytoprotection by global SUMOylation remains undefined. These results support efforts to further augment global protein SUMOylation in brain ischemia.

Thalamic Involvement and Its Impact on Clinical Disability in Patients with Multiple Sclerosis: A Diffusion Tensor Imaging Study at 3T

BACKGROUND AND PURPOSE: Several studies suggest that grey matter involvement may play a role in multiple sclerosis (MS) pathology. Diffusion tensor imaging (DTI) at 3T was used to investigate the presence of damage to the normal-appearing thalamus in MS and its relationship with disability. MATERIALS AND METHODS: Twenty-four patients with relapsing-remitting (RR, n = 13, age = 41.7 ± 6.1, Expanded Disability Status Scale [EDSS] score = 2.2 ± 1.2) and secondary-progressive (n = 11, age = 46.9 ± 9.6, EDSS = 5.9 ± 1.0) MS and 24 age- and sex-matched healthy volunteers were studied. Fractional anisotropy (FA) and mean diffusivity (MD) were measured in regions of interest of normal-appearing thalamus. We examined group differences in MD and FA and correlations between DTI-derived metrics and clinical or imaging measures of disease. RESULTS: Patients with MS had higher thalamic FA (P < .0001) and MD (P = .035) than volunteers. MD values correlated with the Paced Auditory Serial Addition Task (r = −0.43, P = .034) and motor EDSS (r = 0.47, P = .021) scores. In patients with RRMS, MD values correlated with global EDSS (r = 0.75, P = .003) and motor EDSS (r = 0.68, P = .010). Correlations were found between MD values and T1 and T2 lesion load (r = 0.58, P < .05) and brain parenchymal fraction (r = −0.46, P < .05). CONCLUSIONS: DTI was able to detect abnormalities in normal-appearing thalamus of patients with MS. The strength of association between thalamic DTI measures and functional impairment was in the same range as those seen with standard MR imaging disease measures. The assessment of the integrity of the thalamus with DTI is a promising metric as a marker of disease for future studies.

Characteristics of the sequence effect in Parkinson's disease.

The sequence effect (SE) in Parkinsons disease (PD) is progressive slowing of sequential movements. It is a feature of bradykinesia, but is separate from a general slowness without deterioration over time. It is commonly seen in PD, but its physiology is unclear. We measured general slowness and the SE separately with a computer‐based, modified Purdue pegboard in 11 patients with advanced PD. We conducted a placebo‐controlled, four‐way crossover study to learn whether levodopa and repetitive transcranial magnetic stimulation (rTMS) could improve general slowness or the SE. We also examined the correlation between the SE and clinical fatigue. Levodopa alone and rTMS alone improved general slowness, but rTMS showed no additive effect on levodopa. Levodopa alone, rTMS alone, and their combination did not alleviate the SE. There was no correlation between the SE and fatigue. This study suggests that dopaminergic dysfunction and abnormal motor cortex excitability are not the relevant mechanisms for the SE. Additionally, the SE is not a component of clinical fatigue. Further work is needed to establish the physiology and clinical relevance of the SE.

Abnormal functional connectivity in focal hand dystonia: Mutual information analysis in EEG†‡§

The aim of the present study was to investigate functional connectivity in focal hand dystonia patients to understand the pathophysiology underlying their abnormality in movement. We recorded EEGs from 58 electrodes in 15 focal hand dystonia patients and 15 healthy volunteers during rest and a simple finger‐tapping task that did not induce any dystonic symptoms. We investigated mutual information, which provides a quantitative measure of linear and nonlinear coupling, in the alpha, beta, and gamma bands. Mean mutual information of all 58 channels and mean of the channels of interest representative of regional functional connectivity over sensorimotor areas (C3, CP3, C4, CP4, FCz, and Cz) were evaluated. For both groups, we found enhanced mutual information during the task compared with the rest condition, specifically in the beta and gamma bands for mean mutual information of all channels, and in all bands for mean mutual information of channels of interest. Comparing the focal hand dystonia patients with the healthy volunteers for both rest and task, there was reduced mutual information in the beta band for both mean mutual information of all channels and mean mutual information of channels of interest. Regarding the properties of the connectivity in the beta band, we found that the majority of the mutual information differences were from linear connectivity. The abnormal beta‐band functional connectivity in focal hand dystonia patients suggests deficient brain connectivity.

Confusing placebo effect with natural history in epilepsy: A big data approach.

For unknown reasons, placebos reduce seizures in clinical trials in many patients. It is also unclear why some drugs showing statistical superiority to placebo in one trial may fail to do so in another. Using Seizuretracker.com, a patient‐centered database of 684,825 seizures, we simulated “placebo” and “drug” trials. These simulations were employed to clarify the sources of placebo effects in epilepsy, and to identify methods of diminishing placebo effects. Simulation 1 included 9 trials with a 6‐week baseline and 6‐week test period, starting at time 0, 3, 6…24 months. Here, “placebo” reduced seizures regardless of study start time. Regression‐to‐the‐mean persisted only for 3 to 6 months. Simulation 2 comprised a 6‐week baseline and then 2 years of follow‐up. Seizure frequencies continued to improve throughout follow‐up. Although the group improved, individuals switched from improvement to worsening and back. Simulation 3 involved a placebo‐controlled “drug” trial, to explore methods of placebo response reduction. An efficacious “drug” failed to demonstrate a significant effect compared with “placebo” (p = 0.12), although modifications either in study start time (p = 0.025) or baseline population reduction (p = 0.0028) allowed the drug to achieve a statistically significant effect compared with placebo. In epilepsy clinical trials, some seizure reduction traditionally attributed to placebo effect may reflect the natural course of the disease itself. Understanding these dynamics will allow future investigations into optimal clinical trial design and may lead to identification of more effective therapies. Ann Neurol 2015;78:329–336