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An MUSC blog
Date: Jul 11, 2016

Predicting speech fluency after stroke. Brain images showign features of damage to grey-matter and white-matter regions of brain, reflecting their importance in predicting speech fluency.

Image Caption: Predicting speech fluency after stroke. These are features of gray-matter cortical regions (left) and white-matter tracts (right), reflecting their importance in predicting speech fluency scores. Regions/connections are marked in red when they strongly influence speech fluency, in blue when their influence is moderate, and are left uncolored when the influence is weak or non-existent. Image used courtesy of Dr. Leonardo Bonilha and Dr. Grigori Yourganov of the Medical University of South Carolina, who own the copyright for the image. Published in the June 22 issue of the Journal of Neuroscience (doi:10.1523/JNEUROSCI.4396-15.2016).

Loss or impairment of the ability to speak is one of the most feared complications of stroke—one faced by about 20% of stroke patients. Language, as one of the most complex functions of the brain, is not seated in a single brain region but involves connections between many regions.

In an article published in the June 22, 2016 issue of the Journal of Neuroscience (doi:10.1523/JNEUROSCI.4396-15.2016), investigators at the Medical University of South Carolina (MUSC) and the University of South Carolina (USC) report that mapping all of the brain’s white matter connections after stroke, in addition to imaging the areas of cortical tissue damage, could better predict which patients will have language deficits and how severe those deficits will be. The totality of the brain’s connections is referred to as the connectome.

“Imaging the connectome of patients after stroke enables the identification of individual signatures of brain organization that can be used to predict the nature and severity of language deficits and one day could be used to guide therapy,” said MUSC Health neurologist Leonardo Bonilha M.D., Ph.D., senior author on the Journal of Neuroscience article, whose laboratory focuses on connectome imaging, particularly as it relates to language loss after stroke. Grigori Yourganov, Ph.D., is the first author on the article. Julius Fridriksson, Ph.D., Chris Rorden, Ph.D., and  Ezequiel Gleichgerrcht, Ph.D, aphasia researchers at USC who recently received NIH funding to establish a Center for the Study of Aphasia Recovery and who are long-time collaborators of the Bonilha laboratory, are also authors on the article.

This study is the one of the first to use whole-brain connectome imaging to examine how disruptions to white matter connectivity after stroke affect language abilities. White matter fiber tracts are the insulated wires that connect one area of the brain to others. White matter is named for the myelin sheaths (insulation) that cover the many axons (wires) that make up the fiber tracts.

“If you have two brain areas and both of them have to work together in order to carry out a function and the stroke lesion takes out axons that connect those brain areas—the two areas are intact but the communication between them is disrupted and so there is dysfunction,” said Yourganov.

Currently, structural magnetic resonance imaging (MRI) is used after stroke to assess lesions in the cortical tissue—the brain’s grey matter. However, the extent of cortical damage often does not correlate with the severity of language deficits.

“Stroke patients sometimes have significant impairments beyond the amount of cortical damage,” said Bonilha. “It is also hard to predict how well a patient will recover based on the cortical lesion alone.”

Could connectome-based imaging be a useful complement for assessing damage to the brain’s connections after stroke and for guiding rehabilitative therapy?

The study led by Bonilha took an important first step toward answering these questions. The study, which enrolled 90 patients at MUSC and USC with aphasia due to a single stroke occurring no less than six months prior, assessed four areas related to speech/language using the Western Aphasia Battery—speech fluency, auditory comprehension, speech repetition, and oral naming—as well as a summary score of overall aphasia. Within two days of behavior assessment, each of the patients underwent imaging studies—both T1- and T2- weighted MRI, typically used after stroke to map cortical damage, and diffusion imaging, used for connectome mapping. The team then used a type of machine learning algorithm—support vector regression (SVR)—to analyze the imaging results and make predictions about each patient’s language deficits.  In essence, an algorithm was created that could derive the WAB score from either a feature relevant to imaging of the grey matter damage by structural MRI or a feature relevant to connectome imaging of the brain’s white matter fiber tracts. The team used 89 of the 90 patients as training sets for SVR and then used the algorithm to predict language defect/preservation in the 90th patient. This was done for each of the 90 patients and, in each patient, for both features identified via structural MRI and connectome imaging.

The accuracy of the algorithm’s prediction of WAB score for each patient was then assessed by comparing it to the WAB score determined via behavioral testing. Connectome-based analysis was as accurate as cortical lesion mapping for predicting WAB scores. In fact, it was better at predicting auditory comprehension scores than was lesion-based imaging using structural MRI and only slightly less accurate at predicting speech fluency, speech repetition, and naming scores.

The study demonstrates that damage to the white matter fiber tracts that connect the brain’s regions plays a role beyond cortical damage in language impairment after stroke. Furthermore, this study also discloses that connections in the brain’s parietal region are particularly important for language function, especially fluency. This region is less likely to sustain damage after stroke, even in patients who experience aphasia, suggesting that damage or preservation of the brain’s connections in this region could play a key role in determining who will experience aphasia and who will have the best chances for recovery. The integrity of these connections could not be mapped with conventional structural MRI but can now be assessed through connectome-based analysis.

The study findings also suggest that connectome-based analysis could be used to inform a more individualized approach to stroke care. Because the algorithms developed using these study patients as the training set are generalizable to a broader stroke population, connectome-based analysis could one day be used to identify the distinctive features of each patient’s stroke—which connections have been lost and which preserved—and then the algorithm could be used to predict the type and severity of language impairment and the potential for recovery. This information could then be used to direct rehabilitative therapy to improve outcomes. 

“By mapping much more accurately the individual pattern of brain structural connectivity in a stroke survivor, we can determine the integrity of neuronal networks and better understand what was lesioned and how that relates to language abilities that are lost,” said Bonilha. “This is, broadly stated, a measure of post-stroke brain health. It is the individual signature pattern that could also be used to inform about the personalized potential for recovery with therapy and guide treatments to focus on the deficient components of the network.”

Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days.










Summary: In an article published online in Nature Cell Biology on July 11, 2016, investigators at the Medical University of South Carolina report preclinical findings suggesting that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis. Maintaining Dab2 by inhibiting cathepsin B could prevent tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

The results of preclinical studies by investigators at the Medical University of South Carolina (MUSC) reported in an article published online on July 11, 2016 in Nature Cell Biology (doi: 10.1038/ncb3388) demonstrate that disabled 2 (Dab2) serves as a molecular switch that regulates whether a tumor cell undergoes autophagy or apoptosis.

While expression of Dab2—an endocytic adaptor and tumor suppressor—is known to occur during transforming growth factor-beta (TGF-beta)–mediated epithelial-mesenchymal transition (EMT), the mechanisms by which it regulates apoptosis were, until now, poorly understood.

Exploring the pathways by which Dab2 is degraded and the effects of maintaining Dab2 levels reveals its pivotal role in preventing tumor cell survival by blocking autophagy and promoting cell death. These insights provide important information for maximizing the efficacy of existing chemotherapeutic agents.

TGF-beta induces EMT—a process by which cells transform from a polarized epithelial phenotype to a fibroblastic or mesenchymal one. Dab2 is expressed during TGF-beta–mediated EMT. While EMT is essential for normal cellular growth and homeostasis, it is abnormally activated in tumor cells and contributes to their chemo-resistance and metastasis.

TGF-beta has also been reported to regulate autophagy, which, in established tumors, ensures tumor cell survival through times of stress, as for example during chemotherapy. In other words, autophagy supports the chemo-resistance, growth, and metastasis of tumor cells.

Researchers focused on the Dab2 protein after noticing that, in cells treated with TGF-beta, Dab2 levels rose over the initial 24-48 hours as they went through EMT but then fell with continued TGF-beta treatment. By day 7, the tumor cells had transitioned to a morphological state suggestive of either autophagy or apoptosis. Furthermore, the mesenchymal markers N-cadherin and vimentin, which like Dab2 initially rose during EMT, began to decline with longer exposure to TGF-beta.

"This was an unexpected finding that we followed,” explains senior author Philip Howe, Ph.D., Professor and Chair of Biochemistry and Molecular Biology and Hans and Helen Koebig Chair in Clinical Oncology at MUSC. “We knew that if you give cells TGF-beta they go through EMT, and we knew you needed Dab2 for TGF-beta–mediated EMT. But, when we kept adding TGF-beta for more sustained periods (after EMT took place), cells took on a different morphology and we noticed a loss of Dab2. We investigated this loss of Dab2 and discovered that it was being cleaved and that the cells were undergoing autophagy. Upon sustained TGF-beta treatment, the cells had lost their mesenchymal phenotype they'd gained in EMT and entered into an autophagic state.”

The team began to explore how prolonged TGF-beta treatment led to loss of Dab2 and the mesenchymal phenotype.

First, they found that longer TGF-beta exposure significantly increased cathepsin B (CTSB) expression and promoted its co-localization with Dab2. The team then not only demonstrated that CTSB is responsible for cleaving Dab2 but also that it recognizes the cleavage site by the flanking amino acids Val499 and Gly500. Thus, while an unaltered Dab2 sequence (Leu-Val-Gly-Leu) was degraded by CTSB, it did not cleave a mutant Dab2 sequence (Leu-Val-Leu).

Second, findings showed that, after 7 days, continuous TGF-beta treatment induced autophagy and down-regulated markers of apoptosis. This was particularly notable because these conditions promote tumor cell chemo-resistance and metastasis.

Third, they found that CTSB inhibition or expression of a mutant Dab2 without the CTSB cleavage site (i.e., the Leu-Val-Leu mutant) led to time-dependent increases in pro-apoptotic markers. When TGF-beta was withdrawn, cells in which Dab2 had been preserved underwent cell death. This series of experiments show not only how Dab2 is modulated by CTSB but also that it serves as a switch for regulating TGF-b–induced autophagy and apoptosis.

Another series of experiments were undertaken to clarify exactly how Dab2 functions to prevent autophagy and promote apoptosis. These findings show that Dab2 inhibits TGF-beta–induced autophagy by blocking the Vps-Beclin-1 interaction and promotes apoptosis by attenuating ERK-Bim interactions.

Finally, the team used the chemotherapeutic agent doxorubicin (DOXO) to determine whether the role of Dab2 in inhibiting autophagy might affect tumor cell chemo-sensitivity. They found that cells in which CTSB was overexpressed had increased survival in the presence of DOXO. However, cells with high Dab2 levels due to CTSB inhibition or expression of the CTSB-resistant Dab2 mutant were more chemo-sensitive and underwent apoptotic changes. Thus, Dab2 was shown to promote chemotherapeutic drug–induced cell death by attenuating drug-induced autophagy. In vivo tumor studies in mice further found that Dab2 both enhanced DOXO-mediated cell death and attenuated tumor cell metastasis.   

These direct insights into molecular mechanisms supporting tumor cell survival and death are crucial for maximizing the effectiveness of existing chemotherapeutic agents. "This is important because there aren't a whole lot of drugs out there," explains Howe. "Most of what we use today has been around for 20 or 30 years because of a lack of investment in basic science." 

The team's next steps are to investigate in vivo models for combination therapies using DOXO and a CTSB inhibitor to further illuminate the potential for targeting Dab2 as a means of reducing tumor recurrence and metastasis.

Image Caption: In the absence of Disabled-2 (Dab2), Vps34/Beclin-1 Interactions are maintained. Immunofluorescence analysis to detect the expression and localization of Vps34 and Beclin-1 in cathepsin B overexpressing mouse mammary epithelial (CTSB/OE cells) treated -/+ TGF-beta for 7 days. Photos were taken by confocal microscope. Scale bars, 10 mm. The data show that in the absence of Dab2, due to CTSB overexpression, Vsp34/Beclin-1 interactions are maintained and autophagy is initiated. Adapted from a figure originally published in an article by Jiang Y, Woosley AN, Sivalingam N, Natarajan S, and Howe PH in Nature Cell Biology (doi: 10.1038/ncb3388).


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