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Archive for the ‘Cell Biology, Signaling & Cell Circuits’ Category


Disentangling molecular alterations from water-content changes in the aging human brain using quantitative MRI

Reporter: Dror Nir, PhD

 

Authors’ list: Shir Filo, Oshrat Shtangel, Noga Salamon, Adi Kol, Batsheva Weisinger, Sagiv Shifman & Aviv A. Mezer
Published in: Nature Communications volume 10, Article number: 3403 (2019)

Abstract

It is an open question whether aging-related changes throughout the brain are driven by a common factor or result from several distinct molecular mechanisms. Quantitative magnetic resonance imaging (qMRI) provides biophysical parametric measurements allowing for non-invasive mapping of the aging human brain. However, qMRI measurements change in response to both molecular composition and water content. Here, we present a tissue relaxivity approach that disentangles these two tissue components and decodes molecular information from the MRI signal. Our approach enables us to reveal the molecular composition of lipid samples and predict lipidomics measurements of the brain. It produces unique molecular signatures across the brain, which are correlated with specific gene-expression profiles. We uncover region-specific molecular changes associated with brain aging. These changes are independent from other MRI aging markers. Our approach opens the door to a quantitative characterization of the biological sources for aging, that until now was possible only post-mortem.

 

Introduction

The biology of the aging process is complex, and involves various physiological changes throughout cells and tissues1. One of the major changes is atrophy, which can be monitored by measuring macroscale brain volume reduction1,2. In some cases, atrophy can also be detected as localized microscale tissue loss reflected by increased water content3. This process is selective for specific brain regions and is thought to be correlated with cognitive decline in Alzheimer’s disease2,4,5. In addition to atrophy, there are molecular changes associated with the aging of both the normal and pathological brain5,6. Specifically, lipidome changes are observed with age, and are associated with several neurological diseases7,8,9,10,11.

It is an open question as to whether there are general principles that govern the aging process, or whether each system, tissue, or cell deteriorates with age for different reasons12,13. On one hand, the common-cause hypothesis proposes that different biological aging-related changes are the result of a single underlying factor14,15. This implies that various biomarkers of aging will be highly correlated16. On the other hand, the mosaic theory of aging suggests that there are several distinct aging mechanisms that have a heterogenous effect throughout the brain12,13. According to this latter view, combining different measurements of brain tissue is crucial in order to fully describe the state of the aging brain. To test these two competing hypotheses in the context of volumetric and molecular aging-related changes, it is essential to measure different biological aspects of brain tissue. Unfortunately, the molecular correlates of aging are not readily accessible by current in vivo imaging methods.

The main technique used for non-invasive mapping of the aging process in the human brain is magnetic resonance imaging (MRI)2,17,18,19. Advances in the field have led to the development of quantitative MRI (qMRI). This technique provides biophysical parametric measurements that are useful in the investigation and diagnosis of normal and abnormal aging20,21,22,23,24,25,26,27. qMRI parameters have been shown to be sensitive to the microenvironment of brain tissue and are therefore named in vivo histology28,29,30. Nevertheless, an important challenge in applying qMRI measurements is increasing their biological interpretability. It is common to assume that qMRI parameters are sensitive to the myelin fraction20,23,30,31,32,33, yet any brain tissue including myelin is a mixture of multiple lipids and proteins. Moreover, since water protons serve as the source of the MRI signal, the sensitivity of qMRI parameters to different molecular microenvironments may be confounded by their sensitivity to the water content of the tissue34,35. We hypothesized that the changes observed with aging in MRI measurements20,23,30,31,32,33,36 such as R1, R2, mean diffusivity (MD), and magnetization transfer saturation (MTsat)37, could be due to a combination of an increase in water content at the expense of tissue loss, and molecular alterations in the tissue.

Here, we present a qMRI analysis that separately addresses the contribution of changes in molecular composition and water content to brain aging. Disentangling these two factors goes beyond the widely accepted “myelin hypothesis” by increasing the biological specificity of qMRI measurements to the molecular composition of the brain. For this purpose, we generalize the concept of relaxivity, which is defined as the dependency of MR relaxation parameters on the concentration of a contrast agent38. Instead of a contrast agent, our approach exploits the qMRI measurement of the local non-water fraction39 to assess the relaxivity of the brain tissue itself. This approach allows us to decode the molecular composition from the MRI signal. In samples of known composition, our approach provides unique signatures for different brain lipids. In the live human brain, it produces unique molecular signatures for different brain regions. Moreover, these MRI signatures agree with post-mortem measurements of the brain lipid and macromolecular composition, as well as with specific gene-expression profiles. To further validate the sensitivity of the relaxivity signatures to molecular composition, we perform direct comparison of MRI and lipidomics on post-mortem brains. We exploit our approach for multidimensional characterization of aging-related changes that are associated with alterations in the molecular composition of the brain. Finally, we evaluate the spatial pattern of these changes throughout the brain, in order to compare the common-cause and the mosaic theories of aging in vivo.

 

Results

Different brain lipids have unique relaxivity signatures
The aging process in the brain is accompanied by changes in the chemophysical composition, as well as by regional alterations in water content. In order to examine the separate pattern of these changes, we developed a model system. This system was based on lipid samples comprising common brain lipids (phosphatidylcholine, sphingomyelin, phosphatidylserine, phosphatidylcholine-cholesterol, and phosphatidylinositol-phosphatidylcholine)7. Using the model system, we tested whether accounting for the effect of the water content on qMRI parameters provides sensitivity to fine molecular details such as the head groups that distinguish different membrane phospholipids. The non-water fraction of the lipid samples can be estimated by the qMRI measurement of lipid and macromolecular tissue volume (MTV, for full glossary of terms see Supplementary Table 1)39. By varying the concentration of the lipid samples, we could alter their MTV and then examine the effect of this manipulation on qMRI parameters. The parameters we estimated for the lipid samples were R1, R2, and MTsat. The potential ambiguity in the biological interpretation of qMRI parameters is demonstrated in Fig. 1a. On one hand, samples with similar lipid composition can present different R1 measurements (Fig. 1a, points 1 & 2). On the other hand, scanning samples with different lipid compositions may result in similar R1 measurements (Fig. 1a, points 2 & 3). This ambiguity stems from the confounding effect of the water content on the MR relaxation properties.

Screenshot 2019-08-01 at 14.36.20

We evaluated the dependency of different qMRI parameters on the non-water fraction estimated by MTV. This analysis revealed strong linear dependencies (median R2 = 0.74, Fig. 1a, b and Supplementary Fig. 1a, b). These linear MTV dependencies change as a function of the lipid composition, reflecting the inherent relaxivity of the different lipids. We could therefore use the MTV derivatives of qMRI parameters (dqMRIdMTV, i.e., the slope of the linear relationship between each qMRI parameter and MTV) as a measure that is sensitive to molecular composition. By accounting for the Multidimensional Dependency on MTV (“MDM”) of several qMRI parameters, a unique MRI relaxivity signature was revealed for each lipid (Fig. 1c). This implies that the water-related ambiguity demonstrated in the inset of Fig. 1a can be removed by measuring the MTV dependencies (Fig. 1c). Creating mixtures of several lipids provided supportive evidence for the generality of our framework. Figure 1d and Supplementary Fig. 1c show that the qMRI measurements of a mixture can be predicted by summing the MTV dependencies of pure lipids (for further details see Supplementary Note 1 and Supplementary Fig. 2). Furthermore, we used this biophysical model to predict the lipid composition of a mixture from its MDM measurements (Fig. 1e). This model provided a good estimation of the sphingomyelin (Spg) and phosphatidylserine (PS) content (R2 > 0.64) but failed to predict phosphatidylcholine (PtdCho) content (for further details see Supplementary Note 2). While lipids are considered to be a major source of the MRI signal in the brain 40,41,42,43,44,45, our approach can be applied to other compounds to reveal differences in the MRI signal between different proteins, sugars, and ions (Supplementary Fig. 1d). Hence, the relationships between qMRI parameters and MTV account for the effect of water on MRI measurements and could be of use in quantifying the biological and molecular contributions to the MRI signal of water protons.

The tissue relaxivity of the human brain is region-specific.
In order to target age-related changes in molecular composition, we applied the same approach for the human brain (Fig. 2a).

Screenshot 2019-08-01 at 14.41.35

We found that the linear dependency of qMRI parameters on MTV is not limited to in vitro samples and a similar relationship was also evident in the human brain (Fig. 2b and Supplementary Figs. 3–5). Importantly, different brain regions displayed a distinct dependency on MTV. Therefore, the relaxivity of brain tissue is region-specific. Figure 2b provides an example for the regional linear trends of R1 and MTsat in a single subject. Remarkably, while the thalamus and the pallidum presented relatively similar R1 dependencies on MTV, their MTsat dependencies were different (p < 0.001, two-sample t-test). Compared to these two brain regions, frontal white-matter demonstrated different dependencies on MTV (p < 0.001, two-sample t-test). A better separation between brain regions can therefore be achieved by combining the MTV dependencies of several qMRI parameters (MTsat, MD, R1 and R2). The MTV derivatives of qMRI parameters are consistent across subjects (Fig. 2c and Supplementary Fig. 6), with good agreement between hemispheres (Supplementary Fig. 5). Moreover, they provide a novel pattern of differentiation between brain regions, which is not captured by conventional qMRI methods (Supplementary Fig. 7). In our lipid sample experiments, the MDM approach revealed unique relaxivity signatures of different lipids (Fig. 1c). Therefore, we attribute the observed diversity in the MTV derivatives of qMRI parameters across brain regions to the intrinsic heterogeneity in the chemophysical microenvironment of these regions. The multidimensional dependency of various qMRI parameters on MTV can be represented by the space of MTV derivatives to reveal a unique chemophysical MDM signature for different brain regions (Fig. 2d, see explanatory scheme of the MDM method in Supplementary Fig. 8). Fig. 2 figure2 The MDM method provides region-specific signatures in the in vivo human brain. a Representative MTV, MTsat, and R1 maps. b Calculating the MDM signatures. The dependency of R1 (left) and MTsat (right) on MTV in three brain regions of a single subject. For each region, MTV values were pooled into bins (dots are the median of each bin; shaded area is the median absolute deviation), and a linear fit was calculated (colored lines). The slopes of the linear fit represent the MTV derivatives of R1 and MTsat and vary across brain regions. c The reliability of the MDM method across subjects. Variation in the MTV derivatives of R1 (left) and MTsat (right) in young subjects (N = 23). Different colors represent 14 brain regions (see legend). Edges of each box represent the 25th, and 75th percentiles, median is in black, and whiskers extends to extreme data points. Different brain regions show distinct MTV derivatives. d Unique MDM signatures for different brain regions (in different colors). Each axis is the MTV derivative (“MDM measurements”) of a different qMRI parameter (R1, MTsat, R2, and MD). The range of each axis is in the legend. Colored traces extend between the MDM measurements, shaded areas represent the variation across subjects (N = 23). An overlay of all MDM signatures is marked with dashed lines Full size image The in vivo MDM approach captures ex vivo molecular profiles To validate that the MDM signatures relate to the chemophysical composition of brain tissue, we compared them to a previous study that reported the phospholipid composition of the human brain7. First, we established the comparability between the in vivo MRI measurements and the reported post-mortem data. MTV measures the non-water fraction of the tissue, a quantity that is directly related to the total phospholipid content. Indeed, we found good agreement between the in vivo measurement of MTV and the total phospholipid content across brain regions (R2 = 0.95, Fig. 3a). Söderberg et al.7 identified a unique phospholipid composition for different brain regions along with diverse ratios of phospholipids to proteins and cholesterol. We compared this regional molecular variability to the regional variability in the MDM signatures. To capture the main axes of variation, we performed principal component analysis (PCA) on both the molecular composition of the different brain regions and on their MDM signatures. For each of these two analyses, the first principal component (PC) explained >45% of the variance. The regional projection on the first PC of ex vivo molecular composition was highly correlated (R2 = 0.84, Fig. 3b) with the regional projection on the first PC of in vivo MDM signatures. This confirms that brain regions with a similar molecular composition have similar MDM. Supplementary Fig. 9a provides the correlations of individual lipids with MDM. Importantly, neither MTV nor the first PC of standard qMRI parameters was as strongly correlated with the ex vivo molecular composition as the MDM (Supplementary Fig. 9b, c). We next used the MDM measurements as predictors for molecular properties of different brain regions. Following our content predictions for lipids samples (Fig. 1e), we constructed a weighted linear model for human data (for further details see Supplementary Note 3). To avoid over fitting, we reduced the number of fitted parameters by including only the MDM and the molecular features that accounted for most of the regional variability. The MTV derivatives of R1 and MTsat accounted for most of the variance in MDM. Thus, we used these parameters as inputs to the linear model, while adjusting their weights through cross validation. We tested the performance of this model in predicting the three molecular features that account for most of the variance in the ex vivo molecular composition. Remarkably, MRI-driven MDM measurements provided good predictions for the regional sphingomyelin composition (R2 = 0.56, p < 0.05 for the F-test, Fig. 3c) and the regional ratio of phospholipids to proteins (R2 = 0.56, p < 0.05 for the F-test, Fig. 3c).

Screenshot 2019-08-01 at 14.44.06
Last, we compared the cortical MDM signatures to a gene co-expression network based on a widespread survey of gene expression in the human brain46. Nineteen modules were derived from the gene network, each comprised of a group of genes that co-varies in space. Six out of the nineteen gene modules were significantly correlated with the first PC of MDM. Interestingly, the first PC of MDM across the cortex was correlated most strongly with the two gene modules associated with membranes and synapses (Fig. 4, for further details see Supplementary Note 4 and Supplementary Figs. 10 and 11).

Screenshot 2019-08-01 at 14.47.04

Post-mortem validation for the lipidomic sensitivity of MDM.
The aforementioned analyses demonstrate strong agreement between in vivo MDM measurements and ex vivo molecular composition based on a group-level comparison of two different datasets. Strikingly, we were able to replicate this result at the level of the single brain. To achieve this we performed MRI scans (R1, MTsat, R2, MD, and MTV mapping) followed by histology of two fresh post-mortem porcine brains (Fig. 5a, b). First, we validated the qMRI estimation of MTV using dehydration techniques. MTV values estimated using MRI were in agreement with the non-water fraction found histologically (adjusted R2 = 0.64, p < 0.001 for the F-test, Fig. 5c).

Screenshot 2019-08-01 at 14.50.12
Next, we estimated the lipid composition of different brain regions. Thin-layer chromatography (TLC) was employed to quantify seven neutral and polar lipids (Supplementary Table 2 and Supplementary Fig. 12a). In accordance with the analysis in Fig. 3, we performed PCA to capture the main axes of variation in lipidomics, standard qMRI parameters, and MDM. Figure 5d shows that MTV did not correlate with the molecular variability across the brain, estimated by the 1st PC of lipidomics. Likewise, the molecular variability did not agree with the 1st PC of standard qMRI parameters (Fig. 5e).

Last, we applied the MDM approach to the post-mortem porcine brain. Similar to the human brain, different porcine brain regions have unique MDM signatures (Fig. 5f, g and Supplementary Fig. 12b). Remarkably, we found that agreement between lipid composition and MRI measurements emerges at the level of the MDM signatures. The molecular variability across brain regions significantly correlated with the regional variability in the MDM signatures (adjusted R2 = 0.3, p < 0.01 for the F-test, Fig. 5h). Excluding from the linear regression five outlier brain regions where the histological lipidomics results were 1.5 standard deviations away from the center yielded an even stronger correlation between MDM signatures and lipid composition (adjusted R2 = 0.55, p < 0.001 for the F-test, Supplementary Fig. 12c). This post-mortem analysis validates that the MDM approach allows us to capture molecular information using MRI at the level of the individual brain.

Disentangling water and molecular aging-related changes.
After establishing the sensitivity of the MDM signatures to the molecular composition of the brain, we used them to evaluate the chemophysical changes of the aging process. To assess aging-related changes across the brain, we scanned younger and older subjects (18 older adults aged 67 ± 6 years and 23 younger adults aged 27 ± 2 years). First, we identified significant molecular aging-related changes in the MDM signatures of different brain regions (Figs. 6 and 7, right column; Supplementary Fig. 13). Next, we tested whether the changes in MRI measurements, observed with aging, result from a combination of changes in the molecular composition of the tissue and its water content. We found that although it is common to attribute age-related changes in R1 and MTsat to myelin28,30,36, these qMRI parameters combine several physiological aging aspects. For example, using R1 and MTsat we identified significant aging-related changes in the parietal cortex, the thalamus, the parietal white-matter and the temporal white-matter (Figs. 6 and 7, left column). However, the MDM approach revealed that these changes have different biological sources (Figs. 6 and 7, middle columns; see Supplementary Figs. 14–17 for more brain regions).

Screenshot 2019-08-01 at 14.51.53

Screenshot 2019-08-01 at 14.54.44

Screenshot 2019-08-01 at 14.56.06

In agreement with the mosaic hypothesis, we identified distinct aging patterns for different brain regions. For example, in the hippocampus we found a change in R2* values related to a higher iron concentration with age, along with significant reduction in the total hippocampal volume (Fig. 8a). This age-related shrinkage was not accompanied by lower MTV values, indicating conserved tissue density (Fig. 7b). In addition, there was no significant difference in the hippocampal MDM signature with age (Fig. 7b). Cortical gray-matter areas also exhibited similar trends of volume reduction without major loss in tissue density (Fig. 8a). Unlike the gray matter, in the white matter we did not find volume reduction or large iron accumulation with age (Fig. 8a). However, we did find microscale changes with age in tissue composition, as captured by the MDM signature (Figs. 6a and 7c, and Supplementary Fig. 13), accompanied by a significant density-related decline in MTV (Fig. 8a). These findings are consistent with previous histological studies49,50,51 (see Discussion), and provide the ability to monitor in vivo the different components of the aging mosaic.

Last, to test whether the different biological aging trajectories presented in Fig. 8a share a common cause, we evaluated the correlations between them (Fig. 8b). Importantly, the chemophysical trajectory did not correlate significantly with the iron or volume aging patterns. The spatial distribution of water-related changes was found to correlate with iron content alterations (R2 = 0.27) and chemophysical alterations (R2 = 0.25). However, the strongest correlation between aging-related changes was found in volume and iron content (R2 = 0.77). As shown previously, this correlation may be explained to some extent by a systematic bias in automated tissue classification23. Additional analysis revealed that the different dimensions of the MDM signature capture distinct patterns of aging-related changes (Supplementary Fig. 30). Hence, complementary information regarding the various chemophysical mechanisms underlying brain aging could be gained by combining them.

 

Discussion

Normal brain aging involves multiple changes, at both the microscale and macroscale level. MRI is the main tool for in vivo evaluation of such age-related changes in the human brain. Here, we propose to improve the interpretation of MRI findings by accounting for the fundamental effect of the water content on the imaging parameters. This approach allows for non-invasive mapping of the molecular composition in the aging human brain.

Our work is part of a major paradigm shift in the field of MRI toward in vivo histology30,36,52. The MDM approach contributes to this important change by providing a hypothesis-driven biophysical framework that was rigorously developed. We demonstrated the power of our framework, starting from simple pure lipid phantoms to more complicated lipid mixtures, and from there, to the full complexity of the brain. In the brain, we show both in vivo and post-mortem validations for the molecular sensitivity of the MDM signatures. Early observations relate different qMRI parameters to changes in the fraction of myelin20,23,30,31,32,33,36. The current approach enriches this view and provides better sensitivity to the molecular composition and fraction of myelin and other cellular tissues.

We developed a unique phantom system of lipid samples to validate our method. While the phantom system is clearly far from the complexity of brain tissue, its simplicity allowed us to verify the specificity of our method to the chemophysical environment. Remarkably, our approach revealed unique signatures for different lipids, and is therefore sensitive even to relatively subtle details that distinguish one lipid from another. We chose to validate our approach using membrane lipids based on previous experiments40,41,42,43,44,45. Nevertheless, we do acknowledge the fact that brain tissue comprises many other compounds beside lipids, such as proteins, sugars, and ions. As we have shown, these other compounds also exhibit unique dependency on MTV. The effect of such compounds, along with other factors such as microstructure, and multi-compartment organization28 is probably captured when we apply the MDM approach to the in vivo human brain. Therefore, the phantoms were made to examine the MRI sensitivity for the chemophysical environment, and the human brain data was used to measure the true biological effects in a complex in vivo environment.

Our relaxivity approach captures the molecular signatures of the tissue, but is limited in its abilities to describe the full complexity of the chemophysical environment of the human brain. For example, R1 and R2, which are used to generate the MDM signatures, are also sensitive to the iron content23,48,52. However, we found that most of our findings cannot be attributed to alterations in iron content as measured with R2* (for more details see Supplementary Note 5). While there is great importance in further isolating different molecular components, we argue that accounting for the major effect of water on qMRI parameters (for R2 distributions see Supplementary Fig. 5) is a crucial step towards more specific qMRI interpretation.

We provide evidence from lipids samples and post-mortem data for the sensitivity of the MDM signatures to the molecular environment (Figs. 1e, 3b, and 5h). The variability of MDM values between human brain regions also correlated with specific gene-expression profiles (Fig. 4). While the comparison of in vivo human brain measurements to previously published ex vivo findings is based on two different datasets, these measurements are highly stable across normal subjects and the intersubject variabilities are much smaller than the regional variability. The agreement between the modalities provides strong evidence for the ability of our method to capture molecular information.

Remarkably, we were able to demonstrate the sensitivity of MDM signatures to lipid composition using direct comparison on post-mortem porcine brains. Even though there are many challenges in scanning post-mortem tissue, segmenting it, and comparing it to anatomically relevant histological results, we were able to replicate our in vivo findings. We provide histological validation for the MRI estimation of MTV. Moreover, we find that while standard qMRI parameters and MTV do not explain the lipidomic variability across the brain, the MDM signatures are in agreement with histological results. Lipids constitute the majority of the brain’s dry weight and are known to be important for maintaining neural conduction and chemical balance53,54. The brain lipidome was shown to have a great deal of structural and functional diversity and was found to vary according to age, gender, brain region, and cell type55. Disruptions of the brain lipid metabolism have been linked to different disorders, including Alzheimer’s disease, Parkinson’s disease, depression, and anxiety7,8,11,54,55,56,57. Our results indicate that the MDM approach enhances the consistency between MRI-driven measurements and lipidomics, compared with standard qMRI parameters.

The simplicity of our model, which is based on a first-order approximation of qMRI dependencies, has great advantages in the modeling of complex environments. Importantly, we used lipids samples to show that the contributions of different mixture-components can be summed linearly (Fig. 1d). For contrast agents, the relaxivity is used to characterize the efficiency of different agents. Here, we treated the tissue itself, rather than a contrast material, as an agent to compute the relaxivity of the tissue. While relaxivity is usually calculated for R1 and R2, we extended this concept to other qMRI parameters. Our results showed that the tissue relaxivity changes as a function of the molecular composition. This suggests that the relaxivity of the tissue relates to the surface interaction between the water and the chemophysical environment. A theoretical formulation for the effect of the surface interaction on proton relaxation has been proposed before58,59. Specifically, a biophysical model for the linear relationship between R1 and R2 to the inverse of the water content (1/WC = 1/(1 – MTV)) was suggested by Fullerton et al.43. Interestingly, 1/WC varies almost linearly with MTV in the physiological range of MTV values. Applying our approach with 1/WC instead of MTV produces relatively similar results (Supplementary Fig. 28). However, using MTV as a measure of tissue relaxivity allowed us to generalize the linear model to multiple qMRI parameters, thus producing multidimensional MDM signatures.

We show that the MDM signatures allow for better understanding of the biological sources for the aging-related changes observe with MRI. Normal brain aging involves multiple changes, at both the microscale and macroscale levels. Measurements of macroscale brain volume have been widely used to characterize aging-associated atrophy. Our method of analysis can complement such findings and provide a deeper understanding of microscale processes co-occurring with atrophy. Moreover, it allows us to test whether these various microscale and macroscale processes are caused by a common factor or represent the aging mosaic. Notably, we discovered that different brain regions undergo different biological aging processes. Therefore, combining several measurements of brain tissue is crucial in order to fully describe the state of the aged brain. For example, the macroscale aging-related volume reduction in cortical gray areas was accompanied by conserved tissue density, as estimated by MTV, and region-specific chemophysical changes, as estimated by the MDM. In contrast, in white-matter areas both MDM and MTV changed with age. These microscale alterations were not accompanied by macroscale volume reduction. Our in vivo results were validated by previous histological studies, which reported that the cortex shrinks with age, while the neural density remains relatively constant49,50. In contrast, white matter was found to undergo significant loss of myelinated nerve fibers during aging51. In addition, we found that the shrinkage of the hippocampus with age is accompanied with conserved tissue density and chemophysical composition. This is in agreement with histological findings, which predict drastic changes in hippocampal tissue composition in neurological diseases such as Alzheimer, but not in normal aging49,50,60,61. In contrast, hippocampal macroscale volume reduction was observed in both normal and pathological aging2.

It should be noted that most of the human subjects recruited for this study were from the academic community. However, the different age groups were not matched for variables such as IQ and socioeconomic status. In addition, the sample size in our study was quite small. Therefore, the comparison we made between the two age groups may be affected by variables other than age. Our approach may benefit from validation based on larger quantitative MRI datasets27,62. Yet, we believe we have demonstrated the potential of our method to reveal molecular alterations in the brain. Moreover, the agreement of our findings with previous histological aging studies supports the association between the group differences we measured and brain aging. Our results suggest that the MDM approach may be very useful in differentiating the effects of normal aging from those of neurodegenerative diseases. There is also great potential for applications in other brain research fields besides aging. For example, our approach may be used to advance the study and diagnosis of brain cancer, in which the lipidomic environment undergoes considerable changes63,64,65.

To conclude, we have presented here a quantitative MRI approach that decodes the molecular composition of the aging brain. While common MRI measurements are primarily affected by the water content of the tissue, our method employed the tissue relaxivity to expose the sensitivity of MRI to the molecular microenvironment. We presented evidence from lipid samples, post-mortem porcine brains and in vivo human brains for the sensitivity of the tissue relaxivity to molecular composition. Results obtained by this method in vivo disentangled different biological processes occurring in the human brain during aging. We identified region-specific patterns of microscale aging-related changes that are associated with the molecular composition of the human brain. Moreover, we showed that, in agreement with the mosaic theory of aging, different biological age-related processes measured in vivo have unique spatial patterns throughout the brain. The ability to identify and localize different age-derived processes in vivo may further advance human brain research.

Methods

Phantom construction
The full protocol of lipids phantom preparation is described in Shtangel et al.66.

In short, we prepared liposomes from one of the following lipids: phosphatidylserine (PS), phosphatidylcholine (PtdCho), phosphatidylcholine-cholesterol (PtdCho-Chol), Phosphatidylinositol-phosphatidylcholine (PI-PtdCho), or sphingomyelin (Spg). These phantoms were designed to model biological membranes and were prepared from lipids by the hydration–dehydration dry film technique67. The lipids were dissolved over a hot plate and vortexed. Next, the solvent was removed to create a dry film by vacuum-rotational evaporation. The samples were then stirred on a hot plate at 65 °C for 2.5 h to allow the lipids to achieve their final conformation as liposomes. Liposomes were diluted with Dulbecco’s phosphate buffered saline (PBS), without calcium and magnesium (Biological Industries), to maintain physiological conditions in terms of osmolarity, ion concentrations and pH. To change the MTV of the liposome samples we varied the PBS to lipid volume ratios66. Samples were then transferred to the phantom box for scanning in a 4 mL squared polystyrene cuvettes glued to a polystyrene box, which was then filled with ~1% SeaKem Agarose (Ornat Biochemical) and ~0.0005 M Gd (Gadotetrate Melumine, (Dotarem, Guerbet)) dissolved in double distilled water (ddw). The purpose of the agar with Gd (Agar-Gd) was to stabilize the cuvettes, and to create a smooth area in the space surrounding the cuvettes that minimalized air–cuvette interfaces. In some of our experiments we used lipid mixtures composed of several lipids. We prepared nine mixtures containing different combinations of two out of three lipids (PtdChol, Spg and PS) in varying volume ratios (1:1,1:2,2:1). For each mixture, we prepared samples in which the ratio between the different lipid components remained constant while the water-to-lipid volume fraction varied.

For the bovine serum albumin (BSA) phantoms, samples were prepared by dissolving lyophilized BSA powder (Sigma Aldrich) in PBS. To change the MTV of these phantoms, we changed the BSA concentration. For the BSA + Iron phantoms, BSA was additionally mixed with a fixed concentration of 50 µg/mL ferrous sulfate heptahydrate (FeSO4*7H2O). Samples were prepared in their designated concentrations at room temperature. Prepared samples were allowed to sit overnight at 4 ℃ to ensure BSA had fully dissolved, without the need for significant agitation, which is known to cause protein cross-linking. Samples were then transferred to the phantom box for scanning.

For Glucose and Sucrose phantoms, different concentrations of D-( + )-Sucrose (Bio-Lab) and D-( + )-Glucose (Sigma) were dissolved in PBS at 40 ℃. Samples were allowed to reach room temperature before the scan.

MRI acquisition for phantoms

Data was collected on a 3 T Siemens MAGNETOM Skyra scanner equipped with a 32-channel head receive-only coil at the ELSC neuroimaging unit at the Hebrew University.

For quantitative R1 & MTV mapping, three-dimensional (3D) Spoiled gradient (SPGR) echo images were acquired with different flip angles (α = 4°, 8°, 16°, and 30°). The TE/TR was 3.91/18 ms. The scan resolution was 1.1 × 1.1 × 0.9 mm. The same sequence was repeated with a higher resolution of 0.6 × 0.6 × 0.5 mm. The TE/TR was 4.45/18 ms. For calibration, we acquired an additional spin-echo inversion recovery (SEIR) scan. This scan was done on a single slice, with adiabatic inversion pulse and inversion times of TI = 2000, 1200, 800, 400, and 50. The TE/TR was 73/2540 ms. The scan resolution was 1.2 mm isotropic.

For quantitative T2 mapping, images were acquired with a multi spin-echo sequence with 15 equally spaced spin echoes between 10.5 ms and 157.5 ms. The TR was 4.94 s. The scan resolution was 1.2 mm isotropic. For quantitative MTsat mapping, images were acquired with the FLASH Siemens WIP 805 sequence. The TR was 23 ms for all samples except PI:PtdCho for which the TR was 72 ms. Six echoes were equally spaced between 1.93 ms to 14.58 ms. The on-resonance flip angle was 6°, the MT flip angle was 220°, and the RF offset was 700. We used 1.1-mm in-plane resolution with a slice thickness of 0.9 mm. For samples of sucrose and glucose, MTsat mapping was done similar to the human subjects, based on 3D Spoiled gradient (SPGR) echo image with an additional MT pulse. The flip angle was 10°, the TE/TR was 3.91/28 ms. The scan resolution was 1 mm isotropic.

Estimation of qMRI parameters for phantoms

MTV and R1 estimations for the lipids samples were computed based on a the mrQ39 (https://github.com/mezera/mrQ) and Vista Lab (https://github.com/vistalab/vistasoft/wiki) software. The mrQ software was modified to suit the phantom system66. The modification utilizes the fact that the Agar-Gd filling the box around the samples is homogeneous and can, therefore, be assumed to have a constant T1 value. We used this gold standard T1 value generated from the SEIR scan to correct for the excite bias in the spoiled gradient echo scans. While the data was acquired in two different resolutions (see “MRI acquisition”), in our analysis we use the median R1 and MTV of each lipid sample and these are invariant to the resolution of acquisition (Supplementary Fig. 1e). Thus, we were able to use scans with different resolutions without damaging our results. T2 maps were computed by implementing the echo‐modulation curve (EMC) algorithm68.

For quantitative MTsat mapping see the “MTsat estimation” section for human subjects.

MDM computation for phantoms

We computed the dependency of each qMRI parameter (R1, MTsat, and R2) on MTV in different lipids samples. This process was implemented in MATLAB (MathWorks, Natwick, MI, USA). To manipulate the MTV values, we scanned samples of the same lipid in varying concentrations. We computed the median MTV of each sample, along with the median of qMRI parameters. We used these data points to fit a linear model across all samples of the same lipid. The slope of this linear model represents the MTV derivative of the linear equation. We used this derivative estimate of three qMRI parameters (R1, R2, and MTsat) to compute the MDM signatures. The same procedure was used for the MDM computation of lipid mixtures.

 

MDM modeling of lipid mixtures

We tested the ability of MDM to predict the composition of lipid mixtures. For this analysis we used nine mixture phantoms (see “Phantom construction”), along with the three phantoms of the pure lipid constituents of the mixtures (PS, Spg, and Ptd-Cho).

In order to predict the qMRI parameters of a lipid mixture (Fig. 1d) we used Supplementary Eq. 1 (Supplementary Note 1). To further predict the composition of the mixtures (Fig. 1e) we used Supplementary Eq. 5 (Supplementary Note 2). We solved this equation using the QR factorization algorithm.

Ethics

Human experiments complied with all relevant ethical regations. The Helsinki Ethics Committee of Hadassah Hospital, Jerusalem, Israel approved the experimental procedure. Written informed consent was obtained from each participant prior to the procedure.

Human subjects

Human measurements were performed on 23 young adults (aged 27 ± 2 years, 11 females), and 18 older adults (aged 67 ± 6 years, five females). Healthy volunteers were recruited from the community surrounding the Hebrew University of Jerusalem.

MRI acquisition for human subjects

Data was collected on a 3 T Siemens MAGNETOM Skyra scanner equipped with a 32-channel head receive-only coil at the ELSC neuroimaging unit at the Hebrew University.

For quantitative R1, R2*, & MTV mapping, 3D Spoiled gradient (SPGR) echo images were acquired with different flip angles (α = 4°, 10°, 20°, and 30°). Each image included five equally spaced echoes (TE = 3.34–14.02 ms) and the TR was 19 ms (except for six young subjects for which the scan included only one TE = 3.34 ms). The scan resolution was 1 mm isotropic. For calibration, we acquired additional spin-echo inversion recovery scan with an echo-planar imaging (EPI) read-out (SEIR-epi). This scan was done with a slab-inversion pulse and spatial-spectral fat suppression. For SEIR-epi, the TE/TR was 49/2920 ms. TI were 200, 400, 1,200, and 2400 ms. We used 2-mm in-plane resolution with a slice thickness of 3 mm. The EPI read-out was performed using 2 × acceleration.

For quantitative T2 mapping, multi‐SE images were acquired with ten equally spaced spin echoes between 12 ms and 120 ms. The TR was 4.21 s. The scan resolution was 2 mm isotropic. T2 scans of four subjects (one young, three old) were excluded from the analysis due to motion.

For quantitative MTsat mapping, 3D Spoiled gradient (SPGR) echo image were acquired with an additional MT pulse. The flip angle was 10°, the TE/TR was 3.34/27 ms. The scan resolution was 1 mm isotropic.

Whole-brain DTI measurements were performed using a diffusion-weighted spin-echo EPI sequence with isotropic 1.5-mm resolution. Diffusion weighting gradients were applied at 64 directions and the strength of the diffusion weighting was set to b = 2000 s/mm2 (TE/TR = 95.80/6000 ms, G = 45mT/m, δ = 32.25 ms, Δ = 52.02 ms). The data includes eight non-diffusion-weighted images (b = 0). In addition, we collected non-diffusion-weighted images with reversed phase-encode blips. For five subjects (four young, one old) we failed to acquire this correction data and they were excluded from the diffusion analysis.

Anatomical images were acquired with 3D magnetization prepared rapid gradient echo (MP-RAGE) scans for 24 of the subjects (14 from the younger subjects, 10 from the older subjects). The scan resolution was 1 mm isotropic, the TE/TR was 2.98/2300 ms. Magnetization Prepared 2 Rapid Acquisition Gradient Echoes (MP2RAGE) scans were acquired for the rest of the subjects. The scan resolution was 1 mm isotropic, the TE/TR was 2.98/5000 ms.

Estimation of qMRI parameters for human subjects

Whole-brain MTV and R1 maps, together with bias correction maps of B1 + and B1-, were computed using the mrQ software39,69 (https://github.com/mezera/mrQ). Voxels in which the B1 + inhomogeneities were extrapolated and not interpolated were removed from the MTV and R1 maps. While we did not correct our MTV estimates for R2*, we showed that employing such a correction does not significantly change our results (see Supplementary Note 6, Supplementary Figs. 20–27). MTV maps of four subjects had bias in the lower part of the brain and they were therefore excluded from the analysis presented in Fig. 3, which includes ROIs in the brainstem.

Whole-brain T2 maps were computed by implementing the echo‐modulation curve (EMC) algorithm68. To combine the MTV and T2 we co-registered the quantitative MTV map to the T2 map. We used the ANTS software package70 to calculate the transformation and to warp the MTV map and the segmentation. The registration was computed to match the T1 map to the T2 map. Next, we applied the calculated transformation to MTV map (since MTV and T1 are in the same imaging space) and resampled the MTV map to match the resolution of the T2 map. The same transformation was also applied to the segmentation. R2 maps were calculated as 1/T2.

Whole-brain MTsat maps were computed as described in Helms et al.37. The MTsat measurement was extracted from Eq. (1):

MTsat=𝑀0𝐵1𝛼𝑅1TR𝑆MT−(𝐵1𝛼)22−𝑅1TR
(1)
Where SMT is the signal of the SPGR scan with additional MT pulse, α is the flip angle and TR is the repetition time. Mo (the equilibrium magnetization parameter), B1 (the transmit inhomogeneity), and R1 estimations were computed from the non-MT weighted SPGR scans, during the pipeline described under “MTV & R1 estimation”. Registration of the SMT image to the imaging space of the MTV map was done using a rigid-body alignment (R1, B1, and MO are all in the same space as MTV).

Diffusion analysis was done using the FDT toolbox in FSL71,72. Susceptibility and eddy current induced distortions were corrected using the reverse phase-encode data, with the eddy and topup commands73,74. MD maps were calculated using vistasoft (https://github.com/vistalab/vistasoft/wiki). We used a rigid-body alignment to register the corrected dMRI data to the imaging space of the MTV map (Flirt, FSL). In order to calculate the MD-MTV derivatives, we resampled the MTV map and the segmentation to match the dMRI resolution.

We used the SPGR scans with multiple echoes to estimate R2*. Fitting was done through the MPM toolbox75. As we had four SPGR scans with variable flip angles, we averaged the R2* maps acquired from each of these scans for increased SNR.

Human brain segmentation

Whole-brain segmentation was computed automatically using the FreeSurfer segmentation algorithm76. For subjects who had an MP-RAGE scan, we used it as a reference. For the other subjects the MP2RAGE scan was used as a reference. These anatomical images were registered to the MTV space prior to the segmentation process, using a rigid-body alignment. Sub-cortical gray-matter structures were segmented with FSL’s FIRST tool77. To avoid partial volume effects, we removed the outer shell of each ROI and left only the core.

MDM computation in the human brain

We computed the dependency of each qMRI parameter (R1, MTsat, MD, and R2) on MTV in different brain areas. This process was implemented in MATLAB (MathWorks, Natwick, MI, USA). For each ROI, we extracted the MTV values from all voxels and pooled them into 36 bins spaced equally between 0.05 and 0.40. This was done so that the linear fit would not be heavily affected by the density of the voxels in different MTV values. We removed any bins in which the number of voxels was smaller than 4% of the total voxel count in the ROI. The median MTV of each bin was computed, along with the median of the qMRI parameter. We used these data points to fit the linear model across bins using Eq. (2):

qMRIparameters=𝑎∗MTV+𝑏
(2)
The slope of this linear model (“a”) represents the MTV derivative of the linear equation. We used this derivative estimate to compute the MDM signatures.

For each subject, ROIs in which the total voxel count was smaller than a set threshold of 500 voxels for the MTsat and R1 maps, 150 voxels for the MD map, and 50 voxels for the R2 map were excluded.

Principal component analysis (PCA) in the human brain

To estimate the variability in the MDM signatures across the brain, we computed the first principal component (PC) of MDM. For each MDM dimension (MTV derivatives of R1, MTsat, MD, and R2), we evaluated the median of the different brain areas across the young subjects. As each MDM dimension has different units, we then computed the z-score of each dimension across the different brain area. Finally, we performed PCA. The variables in this analysis were the different MDM dimensions, and the observations were the different brain areas. From this analysis, we derived the first PC that accounts for most of the variability in MDM signatures across the brain. To estimate the median absolute deviations (MAD) across subjects of each MDM measurement in the PC basis, we applied the z-score transformation to the original MAD and then projected them onto the PC basis.

To compute the first PC of standard qMRI parameters we followed the same procedure, but used R1, MTsat, MD, and R2 instead of their MTV derivatives.

For the first PC of molecular composition, we followed the same procedure, but used the phospholipid composition and the ratio between phospholipids to proteins and cholesterol as variables. The data was taken from eight post-mortem human brains7. Brains were obtained from individuals between 54 and 57 years of age, which were autopsied within 24 h after death.

Linear model for prediction of human molecular composition

We used MDM measurements in order to predict the molecular composition of different brain areas (Fig. 3c). For this analysis we used Supplementary Eq. 5 in the Supplementary Note 2. We solved this equation using QR factorization algorithm (for more details see Supplementary Note 3).

Gene-expression dataset

For the gene-expression analysis we followed the work of Ben-David and Shifman46. Microarray data was acquired from the Allen Brain Atlas (http://human.brain-map.org/well_data_files) and included a total of 1340 microarray profiles from donors H0351.2001 and H0351.2002, encompassing the different regions of the human brain. The donors were 24 and 39 years old, respectively, at the time of their death, with no known psychopathologies. We used the statistical analysis described by Ben-David and Shifman46. They constructed a gene network using a weighted gene co-expression network analysis. The gene network included 19 modules of varying sizes, from 38 to 7385 genes. The module eigengenes were derived by taking the first PC of the expression values in each module. In addition, we used the gene ontology enrichment analysis described by Ben-David and Shifman to define the name of each module. The colors of the different modules in the Fig. 4 and Supplementary Fig. 10 are the same as in the original paper.

Next, we matched between the gene-expression data and the MRI measurements. This analysis was done on 35 cortical regions extracted from FreeSurfer cortical parcellation. We downloaded the T1-weighted images of the two donors provided by the Allen Brain Atlas (http://human.brain-map.org/mri_viewers/data) and used them as a reference for FreeSurfer segmentation. We then found the FreeSurfer label of each gene-expression sample using the sample’s coordinates in brain space. We removed samples for which the FreeSurfer label and the label provided in the microarray dataset did not agree (there were 72 such samples out of 697 cortical samples). For each gene module, we averaged over the eigengenes of all samples from the same cortical area across the two donors.

Last, we compared the cortical eigengene of each module to the projection of cortical areas on the first PC of MDM. In addition, we compared the modules’ eigengenes to the MTV values of the cortical areas and to the projection of cortical areas on the first PC of standard qMRI parameters (Supplementary Fig. 10). These 57 correlations were corrected for multiple comparisons using the FDR method.

Brain region’s volume computation

To estimate the volume of different brain regions, we calculated the number of voxels in the FreeSurfer segmentation of each region (see “Brain segmentation”).

R2* correction for MTV
To correct the MTV estimates for R2* we used Eq. (3):

MTV𝐶=1−(1−MTV)⋅exp(TE⋅R2∗)
(3)
Where MTVC is the corrected MTV.

Statistical analysis

The statistical significance of the differences between the age groups was computed using an independent-sample t-test (alpha = 0.05, both right and left tail) and was corrected for multiple comparisons using the false-discovery rate (FDR) method. For this analysis, MRI measurements of both hemispheres of bilateral brain regions were joined together. R2 measurements were adjusted for the number of data points. All statistical tests were two-sided.

Post-mortem tissue acquisition

Two post-mortem porcine brains were purchased from BIOTECH FARM.

Post-mortem MRI acquisition

Brains were scanned fresh (without fixation) in water within 6 h after death. Data was collected on a 3 T Siemens MAGNETOM Skyra scanner equipped with a 32-channel head receive-only coil at the ELSC neuroimaging unit at the Hebrew University.

For quantitative R1, R2*, & MTV mapping, 3D Spoiled gradient (SPGR) echo images were acquired with different flip angles (α = 4°, 10°, 20°, and 30°). Each image included five equally spaced echoes (TE = 4.01 – 16.51 ms) and the TR was 22 ms. The scan resolution was 0.8 mm isotropic. For calibration, we acquired additional spin-echo inversion recovery scan with an echo-planar imaging (EPI) read-out (SEIR-epi). This scan was done with a slab-inversion pulse and spatial-spectral fat suppression. For SEIR-epi, the TE/TR was 49/2920 ms. TI were 50, 200, 400, 1200 ms. The scan resolution was 2 mm isotropic. The EPI read-out was performed using 2 × acceleration.

For quantitative T2 mapping, multi‐SE images were acquired with ten equally spaced spin echoes between 12 and 120 ms. The TR was 4.21 s. The scan resolution was 2 mm isotropic.

For quantitative MTsat mapping, 3D Spoiled gradient (SPGR) echo image were acquired with an additional MT pulse. The flip angle was 10°, the TE/TR was 4.01/40 ms. The scan resolution was 0.8 mm isotropic.

Whole-brain DTI measurements were performed using a diffusion-weighted spin-echo EPI sequence with isotropic 1.5-mm resolution. Diffusion weighting gradients were applied at 64 directions and the strength of the diffusion weighting was set to b = 2000 s/mm2 (TE/TR = 95.80/6000 ms, G = 45mT/m, δ = 32.25 ms, Δ = 52.02 ms). The data includes eight non-diffusion-weighted images (b = 0).

For anatomical images, 3D magnetization prepared rapid gradient echo (MP-RAGE) scans were acquired. The scan resolution was 1 mm isotropic, the TE/TR was 2.98/2300 ms.

Histological analysis

Following the MRI scans the brains were dissected. Total of 42 brain regions were identified. Four samples were excluded as we were not able to properly separate the WM from the GM. One sample was excluded as we could not properly identify its anatomical origin. Additional two samples were too small for TLC analysis.

The non-water fraction (MTV) was determined by desiccation, also known as the dry-wet method. A small fraction of each brain sample (~0.25 g) was weighed. In order to completely dehydrate the fresh tissues, they were left for several days in a vacuum dessicator over silica gel at 4 °C. The experiment ended when no further weight loss occurred. The MTV of each brain sample was calculated based on the difference between the wet (Wwet) and dry (Wdry) weights of the tissue (Eq. 4):

MTV=𝑊wet−𝑊dry𝑊wet
(4)
For lipid extraction and lipidomics analysis78, Brain samples were weighted and homogenized with saline in plastic tubes on ice at concentration of 1 mg/12.5 µL. Two-hundred fifty microliters from each homogenate were utilized for lipid extraction and analysis with thin-layer chromatography (TLC). The lipid species distribution was analyzed by TLC applying 150 µg aliquots. Samples were reconstituted in 10 µL of Folch mixture and spotted on Silica-G TLC plates. Standards for each fraction were purchased from Sigma Aldrich (Rehovot, Israel) and were spotted in separate TLC lanes, i.e., 50 µg of triacylglycerides (TG), cholesterol (Chol), cholesteryl esters (CE), free fatty acids (FFA), lysophospholipids (Lyso), sphingomyelin (Spg), phosphatidylcholine (PtdCho), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Plates were then placed in a 20 × 20 cm TLC chamber containing petroleum ether, ethyl ether, and acetic acid (80:20:1, v/v/v) for quantification of neutral lipids or chloroform, methanol, acetic acid, and water (65:25:4:2, v:v:v:v) for quantification of polar lipids and run for 45 min. TG, Chol, CE, FFA, phospholipids (PL), Lyso, Spg, PtdCho, PI, PS, and PE bands were visualized with Iodine, scanned and quantified by Optiquant after scanning (Epson V700). Lyso, CE, TG, and PI were excluded from further analysis as their quantification was noisy and demonstrated high variability across TLC plates. This analysis was conducted under the guidance of Prof. Alicia Leikin-Frenkel in the Bert Strassburger Lipid Center, Sheba, Tel Hashomer.

Estimation of qMRI parameters in the post-mortem brain

Similar to human subjects.

Brain segmentation of post-mortem brain

Brain segmentation was done manually. Five tissue samples were excluded as we could not identify their origin location in the MRI scans.

MDM computation in the post-mortem brain

We computed the dependency of each qMRI parameter (R1, MTsat, MD, and R2) on MTV in different brain areas similarly to the analysis of the human subjects.

Principal component analysis (PCA) in the post-mortem brain

To estimate the variability in the MDM signatures across the brain, we computed the first principal component (PC) of MDM. PCA analysis was performed with four variables corresponding to the MDM dimensions (MTV derivatives of R1, MTsat, MD, and R2), and 30 observations corresponding to the different brain regions. As each MDM dimension has different units, we first computed the z-score of each dimension across the different brain areas prior to the PCA. From this analysis we derived the first PC that accounts for most of the variability in MDM signatures across the brain.

To compute the first PC of standard qMRI parameters we followed the same procedure, but used R1, MTsat, MD, and R2 instead of their MTV derivatives.

To estimate the variability in the lipid composition across the brain, we computed the first principal component (PC) of lipidomics. PCA analysis was performed with seven variables corresponding to the different polar and neutral lipids (Chol, FFA, PL, Spg, PtdCho, PS, PE), and 30 observations corresponding to the different brain regions. From this analysis, we derived the first PC that accounts for most of the variability in lipid composition across the brain.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

A toolbox for computing MDM signatures is available at [https://github.com/MezerLab/MDM_toolbox].

The code generating the figures of in the paper is available at [https://github.com/MezerLab/MDM_Gen_Figs].

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Acknowledgements

This work was supported by the ISF grant 0399306, awarded to A.A.M. We acknowledge Ady Zelman for the assistance in collecting the human MRI data. We thank Assaf Friedler for assigning research lab space and advising on the lipid sample experiments. We thank Inbal Goshen for assigning research lab space and advising on the protein and ion samples as well as the porcine brain experiments. We thank Magnus Soderberg for advising on histological data interpretation. We are grateful to Brian A. Wandell, Jason Yeatman, Hermona Soreq, Ami Citri, Mark Does, Yaniv Ziv, Ofer Yizhar, Shai Berman, Roey Schurr, Jonathan Bain, Asier Erramuzpe Aliaga, Menachem Gutman, and Esther Nachliel for their critical reading of the manuscript and very useful comments. We thank Prof. Alicia Leikin-Frenkel for her guidance with the TLC analysis. We thank Rona Shaharabani for guidance and support in the post-mortem experiments.

Affiliations

The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University of Jerusalem, Jerusalem, 9190401, Israel
Shir Filo, Oshrat Shtangel, Noga Salamon, Adi Kol, Batsheva Weisinger & Aviv A. Mezer
Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 9190401, Israel
Sagiv Shifman
Contributions
S.F., O.S., and A.A.M. conceived of the presented idea. S.F. and A.A.M. wrote the manuscript and designed the figures. S.F. collected the human and non-human brain datasets and analyzed them. O.S. performed the phantom experiments and analyzed them. B.W. performed the phantom experiments for non-lipid compounds. N.S. performed the gene-expression analysis. S.S. assisted and instructed with the gene-expression analysis. A.K. performed the porcine brain dissection.

Corresponding author

Correspondence to Aviv A. Mezer.

Ethics declarations & Competing interests

A.A.M, S.F., O.S. and the Hebrew University of Jerusalem have filed a patent application describing the technology used to measure MDM in this work. The other authors declare no competing interests.

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An Intelligent DNA Nanorobot to Fight Cancer by Targeting HER2 Expression

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

HER2 is an important prognostic biomarker for 20–30% of breast cancers, which is the most common cancer in women. Overexpression of the HER2 receptor stimulates breast cells to proliferate and differentiate uncontrollably, thereby enhancing the malignancy of breast cancer and resulting in a poor prognosis for affected individuals. Current therapies to suppress the overexpression of HER2 in breast cancer mainly involve treatment with HER2-specific monoclonal antibodies. However, these monoclonal anti-HER2 antibodies have severe side effects in clinical trials, such as diarrhea, abnormal liver function, and drug resistance. Removing HER2 from the plasma membrane or inhibiting the gene expression of HER2 is a promising alternative that could limit the malignancy of HER2-positive cancer cells.

 

DNA origami is an emerging field of DNA-based nanotechnology and intelligent DNA nanorobots show great promise in working as a drug delivery system in healthcare. Different DNA-based nanorobots have been developed as affordable and facile therapeutic drugs. In particular, many studies reported that a tetrahedral framework nucleic acid (tFNA) could serve as a promising DNA nanocarrier for many antitumor drugs, owing to its high biocompatibility and biosecurity. For example, tFNA was reported to effectively deliver paclitaxel or doxorubicin to cancer cells for reversing drug resistance, small interfering RNAs (siRNAs) have been modified into tFNA for targeted drug delivery. Moreover, the production and storage of tFNA are not complicated, and they can be quickly degraded in lysosomes by cells. Since both free HApt and tFNA can be diverted into lysosomes, so,  combining the HApt and tFNA as a novel DNA nanorobot (namely, HApt-tFNA) can be an effective strategy to improve its delivery and therapeutic efficacy in treating HER2-positive breast cancer.

 

Researchers reported that a DNA framework-based intelligent DNA nanorobot for selective lysosomal degradation of tumor-specific proteins on cancer cells. An anti-HER2 aptamer (HApt) was site-specifically anchored on a tetrahedral framework nucleic acid (tFNA). This DNA nanorobot (HApt-tFNA) could target HER2-positive breast cancer cells and specifically induce the lysosomal degradation of the membrane protein HER2. An injection of the DNA nanorobot into a mouse model revealed that the presence of tFNA enhanced the stability and prolonged the blood circulation time of HApt, and HApt-tFNA could therefore drive HER2 into lysosomal degradation with a higher efficiency. The formation of the HER2-HApt-tFNA complexes resulted in the HER2-mediated endocytosis and digestion in lysosomes, which effectively reduced the amount of HER2 on the cell surfaces. An increased HER2 digestion through HApt-tFNA further induced cell apoptosis and arrested cell growth. Hence, this novel DNA nanorobot sheds new light on targeted protein degradation for precision breast cancer therapy.

 

It was previously reported that tFNA was degraded by lysosomes and could enhance cell autophagy. Results indicated that free Cy5-HApt and Cy5-HApt-tFNA could enter the lysosomes; thus, tFNA can be regarded as an efficient nanocarrier to transmit HApt into the target organelle. The DNA nanorobot composed of HApt and tFNA showed a higher stability and a more effective performance than free HApt against HER2-positive breast cancer cells. The PI3K/AKT pathway was inhibited when membrane-bound HER2 decreased in SK-BR-3 cells under the action of HApt-tFNA. The research findings suggest that tFNA can enhance the anticancer effects of HApt on SK-BR-3 cells; while HApt-tFNA can bind to HER2 specifically, the compounded HER2-HApt-tFNA complexes can then be transferred and degraded in lysosomes. After these processes, the accumulation of HER2 in the plasma membrane would decrease, which could also influence the downstream PI3K/AKT signaling pathway that is associated with cell growth and death.

 

However, some limitations need to be noted when interpreting the findings: (i) the cytotoxicity of the nanorobot on HER2-positive cancer cells was weak, and the anticancer effects between conventional monoclonal antibodies and HApt-tFNA was not compared; (ii) the differences in delivery efficiency between tFNA and other nanocarriers need to be confirmed; and (iii) the confirmation of anticancer effects of HApt-tFNA on tumors within animals remains challenging. Despite these limitations, the present study provided novel evidence of the biological effects of tFNA when combined with HApt. Although the stability and the anticancer effects of HApt-tFNA may require further improvement before clinical application, this study initiates a promising step toward the development of nanomedicines with novel and intelligent DNA nanorobots for tumor treatment.

 

References:

 

https://pubs.acs.org/doi/10.1021/acs.nanolett.9b01320

 

https://www.ncbi.nlm.nih.gov/pubmed/27939064

 

https://www.ncbi.nlm.nih.gov/pubmed/11694782

 

https://www.ncbi.nlm.nih.gov/pubmed/27082923

 

https://www.ncbi.nlm.nih.gov/pubmed/25365825

 

https://www.ncbi.nlm.nih.gov/pubmed/26840503

 

https://www.ncbi.nlm.nih.gov/pubmed/29802035

 

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Single-cell RNA-seq helps in finding intra-tumoral heterogeneity in pancreatic cancer

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Pancreatic cancer is a significant cause of cancer mortality; therefore, the development of early diagnostic strategies and effective treatment is essential. Improvements in imaging technology, as well as use of biomarkers are changing the way that pancreas cancer is diagnosed and staged. Although progress in treatment for pancreas cancer has been incremental, development of combination therapies involving both chemotherapeutic and biologic agents is ongoing.

 

Cancer is an evolutionary disease, containing the hallmarks of an asexually reproducing unicellular organism subject to evolutionary paradigms. Pancreatic ductal adenocarcinoma (PDAC) is a particularly robust example of this phenomenon. Genomic features indicate that pancreatic cancer cells are selected for fitness advantages when encountering the geographic and resource-depleted constraints of the microenvironment. Phenotypic adaptations to these pressures help disseminated cells to survive in secondary sites, a major clinical problem for patients with this disease.

 

The immune system varies in cell types, states, and locations. The complex networks, interactions, and responses of immune cells produce diverse cellular ecosystems composed of multiple cell types, accompanied by genetic diversity in antigen receptors. Within this ecosystem, innate and adaptive immune cells maintain and protect tissue function, integrity, and homeostasis upon changes in functional demands and diverse insults. Characterizing this inherent complexity requires studies at single-cell resolution. Recent advances such as massively parallel single-cell RNA sequencing and sophisticated computational methods are catalyzing a revolution in our understanding of immunology.

 

PDAC is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. In the present study to comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, single-cell RNA-seq (scRNA-seq) was employed to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases. The diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, were identified in PDAC.

 

The researchers found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, it was found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, the findings provided a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

 

References:

 

https://www.ncbi.nlm.nih.gov/pubmed/31273297

 

https://www.ncbi.nlm.nih.gov/pubmed/21491194

 

https://www.ncbi.nlm.nih.gov/pubmed/27444064

 

https://www.ncbi.nlm.nih.gov/pubmed/28983043

 

https://www.ncbi.nlm.nih.gov/pubmed/24976721

 

https://www.ncbi.nlm.nih.gov/pubmed/27693023

 

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Celiac Disease Breakthrough: (1) 472 genes regulated differently in organoids reflecting celiac disease than in non-celiac control organoids (2) bio-products derived from gut microorganisms can be employed to modify the epithelial response to gluten, a finding that could lead to future treatment strategies.

 

Reporter: Aviva Lev-Ari, PhD, RN

“These results confirm our hypothesis that genes and exposure to gluten are necessary but not sufficient, since changes in both the composition and function of the gut microbiome are also needed to switch from genetic predisposition to clinical outcome, as shown by our data,” said Alessio Fasano, HMS professor of pediatrics at Mass General, director of MIBRC and co-senior author of the paper.

https://hms.harvard.edu/news/major-shift?utm_source=Silverpop&utm_medium=email&utm_term=field_news_item_3&utm_content=HMNews05132019

 

 

Image Source: iStock/wildpixel

Article OPEN Published: 

Human gut derived-organoids provide model to study gluten response and effects of microbiota-derived molecules in celiac disease

Scientific Reports volume 9, Article number: 7029 (2019Download Citation

Abstract

Celiac disease (CD) is an immune-mediated disorder triggered by gluten exposure. The contribution of the adaptive immune response to CD pathogenesis has been extensively studied, but the absence of valid experimental models has hampered our understanding of the early steps leading to loss of gluten tolerance. Using intestinal organoids developed from duodenal biopsies from both non-celiac (NC) and celiac (CD) patients, we explored the contribution of gut epithelium to CD pathogenesis and the role of microbiota-derived molecules in modulating the epithelium’s response to gluten. When compared to NC, RNA sequencing of CD organoids revealed significantly altered expression of genes associated with gut barrier, innate immune response, and stem cell functions. Monolayers derived from CD organoids exposed to gliadin showed increased intestinal permeability and enhanced secretion of pro-inflammatory cytokines compared to NC controls. Microbiota-derived bioproducts butyrate, lactate, and polysaccharide A improved barrier function and reduced gliadin-induced cytokine secretion. We concluded that: (1) patient-derived organoids faithfully express established and newly identified molecular signatures characteristic of CD. (2) microbiota-derived bioproducts can be used to modulate the epithelial response to gluten. Finally, we validated the use of patient-derived organoids monolayers as a novel tool for the study of CD.

Mass. General researchers develop 3D “mini-gut” model to study autoimmune response to gluten in celiac and non-celiac patient tissue

Gene expression of intestinal organoids reflects functional differences found in celiac disease

In pursuit of a novel tool for the research and treatment of celiac disease, scientists at the Mucosal Immunology and Biology Research Center (MIBRC) at Massachusetts General Hospital (MGH) have validated the use of intestinal organoids. These three-dimensional tissue cultures are miniature, simplified versions of the intestine produced in vitro. Taking tissue from duodenal biopsies of celiac and non-celiac patients, researchers created the “mini-guts” to explore how the gut epithelium and microbiota-derived molecules respond to gluten, a complex class of proteins found in wheat and other grains.

“We currently have no animal model that can recapitulate the response to gluten that we see in humans,” says Stefania Senger, PhD, co-senior author of the study published in Scientific Reports this week. “Using this human tissue model, we observed that intestinal organoids express the same molecular markers as actual epithelium in the celiac tissue, and the signature gene expression reflects the functional differences that occur when epithelia of celiac disease patients are exposed to gliadin.” Gliadin and glutenin proteins are main components of gluten.

Celiac disease is triggered when genetically predisposed individuals consume gluten. The condition affects approximately 1 percent of the U.S. population. Based on current data, the onset of celiac disease is thought to be preceded by the release of the protein zonulin, which is triggered by the activation of undigested gliadin to induce an autoimmune response. This leads to increased intestinal permeability and a disrupted barrier function. Novel evidence suggests that the microorganisms in the gastrointestinal tract may play a role in the onset of celiac disease.

Earlier studies from the MIBRC group and others have shown that human organoids “retain a gene expression that recapitulates the expression of the tissue of origin, including a diseased state,” the authors write. Through RNA sequencing, the new findings validate the organoid model as a “faithful in vitro model for celiac disease,” Senger says.
Using whole-transcriptome analysis, the researchers identified 472 genes regulated differently in organoids reflecting celiac disease than in non-celiac control organoids. These included novel genes associated with epithelial functions related to the pathogenesis of celiac disease – including gut barrier maintenance, stem cell regeneration and innate immune response. A second finding of the study shows that bioproducts derived from gut microorganisms can be employed to modify the epithelial response to gluten, a finding that could lead to future treatment strategies.

“These results confirm our hypothesis that genes and exposure to gluten are necessary but not sufficient, since changes in both the composition and function of the gut microbiome are also needed to switch from genetic predisposition to clinical outcome, as shown by our data,” says Alessio Fasano, MD, director of the Mucosal Immunology and Biology Research Center and co-senior author.

Senger adds, “We believe our observations represent a major shift in the study of celiac disease. We are confident that with adequate funding we could achieve major goals that include the development and implementation of high-throughput drug screenings to quickly identify new treatments for patients and expand the organoid repository to develop more complex models and pursue personalized treatment.”
Additional co-authors of the paper are first author Rachel Freire, PhD, along with Laura Ingano and Gloria Serena, PhD, of the MGH MIBRC; Murat Cetinbas, PhD, and Ruslan Sadreyev, PhD, MGH Department of Molecular Biology; Anthony Anselmo, PhD, formerly of MGH Molecular Biology and now with PatientsLikeMe, Cambridge, Mass.; and Anna Sapone, MD, PhD, Takeda Pharmaceuticals International. Support for the study includes National Institutes of Health grants RO1 DK104344-01A1 and 1U19 AI082655-02 and the Egan Family Foundation.

SOURCE

https://www.massgeneral.org/about/pressrelease.aspx?id=2403

 

Other related articles and e-Books by LPBI Group’s Authors published on this Open Access Online Scientific Journal include the following:

 

Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases

  • Metabolomics 

VOLUME 1: Metabolic Genomics and Pharmaceutics. On Amazon.com since 7/21/2015

http://www.amazon.com/dp/B012BB0ZF0

Gluten-free Diets

Writer and Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2015/03/01/gluten-free-diets/

 

Breakthrough Digestive Disorders Research: Conditions affecting the Gastrointestinal Tract.

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/12/12/breakthrough-digestive-disorders-research-conditions-affecting-the-gastrointestinal-tract/

 

Collagen-binding Molecular Chaperone HSP47: Role in Intestinal Fibrosis – colonic epithelial cells and subepithelial myofibroblasts

Curators: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2016/01/25/collagen-binding-molecular-chaperone-hsp47-role-in-intestinal-fibrosis-colonic-epithelial-cells-and-subepithelial-myofibroblasts/

Expanding area of Tolerance-inducing Autoimmune Disease Therapeutics: Key Players

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2017/01/17/expanding-area-of-tolerance-inducing-autoimmune-disease-therapeutics-key-players/

 

What is the key method to harness Inflammation to close the doors for many complex diseases?

Author and Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Leigh syndrome is one of the hundreds of so-called mitochondrial diseases, which are caused by defects in the mitochondria that produce 90 percent of the body’s energy. These disorders are rare; about 1,000 to 4,000 babies in the United States are born with one every year. But they are devastating and can result in grave impairment of nearly any bodily system. They are largely untreatable, uniformly incurable and very difficult to screen.

 

Leigh syndrome is a terrible disease. It emerges shortly after birth and claims one major organ after another. Movement becomes difficult, and then impossible. A tracheotomy and feeding tube are often necessary by toddlerhood, and as the disease progresses, lungs frequently have to be suctioned manually. Most children with the condition die by the age of 5 or 6.

 

Scientists have devised a procedure called mitochondrial replacement therapy (M.R.T.) that involves transplanting the nucleus of an affected egg (mitochondrial diseases are passed down from the mother’s side) into an unaffected one whose nucleus has been removed. The procedure is sometimes called “three-parent in vitro fertilization”. Mitochondria contain a minuscule amount of DNA, any resulting embryo would have mitochondrial DNA from the donor egg and nuclear DNA from each of its parents.

 

After decades of careful study in cell and animal research M.R.T. is now finally being tested in human clinical trials by doctors in Britain (no births confirmed yet officially). In the United States, however, this procedure is effectively illegal. M.R.T. does not involve altering any genetic code. Defective mitochondria are swapped out for healthy ones.

 

Mitochondrial DNA governs only a handful of basic cellular functions. It is separate from nuclear DNA, which helps determine individual traits like physical appearance, intelligence and personality. That means M.R.T. cannot be used to produce the genetically enhanced “designer babies” and thus should be allowed in humans. But, there is no way to know how safe or effective M.R.T. is until doctors and scientists test it in humans.

 

References:

 

 

https://pharmaceuticalintelligence.com/2016/10/07/the-three-parent-technique-to-avoid-mitochondrial-disease-in-embryo/

 

 

 

 

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Lesson 8 Cell Signaling and Motility: Lesson and Supplemental Information on Cell Junctions and ECM: #TUBiol3373

Curator: Stephen J. Williams, Ph.D.

Please click on the following link for the PowerPoint Presentation for Lecture 8 on Cell Junctions and the  Extracellular Matrix: (this is same lesson from 2018 so don’t worry that file says 2018)

cell signaling 8 lesson 2018

 

Some other reading on this lesson on this Open Access Journal Include:

On Cell Junctions:

Translational Research on the Mechanism of Water and Electrolyte Movements into the Cell     

(pay particular attention to article by Fischbarg on importance of tight junctions for proper water and electrolyte movement)

The Role of Tight Junction Proteins in Water and Electrolyte Transport

(pay attention to article of role of tight junction in kidney in the Loop of Henle and the collecting tubule)

EpCAM [7.4]

(a tight junction protein)

Signaling and Signaling Pathways

(for this lesson pay attention to the part that shows how Receptor Tyrosine Kinase activation (RTK) can lead to signaling to an integrin and also how the thrombin receptor leads to cellular signals both to GPCR (G-protein coupled receptors like the thrombin receptor, the ADP receptor; but also the signaling cascades that lead to integrin activation of integrins leading to adhesion to insoluble fibrin mesh of the newly formed clot and subsequent adhesion of platelets, forming the platelet plug during thrombosis.)

On the Extracellular Matrix

Three-Dimensional Fibroblast Matrix Improves Left Ventricular Function Post MI

Arteriogenesis and Cardiac Repair: Two Biomaterials – Injectable Thymosin beta4 and Myocardial Matrix Hydrogel

 

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Protein kinase C (PKC) isozymes function as tumor suppressors in increasing contexts. These enzymes are crucial for a number of cellular activities, including cell survival, proliferation and migration — functions that must be carefully controlled if cells get out of control and form a tumor. In contrast to oncogenic kinases, whose function is acutely regulated by transient phosphorylation, PKC is constitutively phosphorylated following biosynthesis to yield a stable, autoinhibited enzyme that is reversibly activated by second messengers. Researchers at University of California San Diego School of Medicine found that another enzyme, called PHLPP1, acts as a “proofreader” to keep careful tabs on PKC.

 

The researchers discovered that in pancreatic cancer high PHLPP1 levels lead to low PKC levels, which is associated with poor patient survival. They reported that the phosphatase PHLPP1 opposes PKC phosphorylation during maturation, leading to the degradation of aberrantly active species that do not become autoinhibited. They discovered that any time an over-active PKC is inadvertently produced, the PHLPP1 “proofreader” tags it for destruction. That means the amount of PHLPP1 in patient’s cells determines his amount of PKC and it turns out those enzyme levels are especially important in pancreatic cancer.

 

This team of researchers reversed a 30-year paradigm when they reported evidence that PKC actually suppresses, rather than promotes, tumors. For decades before this revelation, many researchers had attempted to develop drugs that inhibit PKC as a means to treat cancer. Their study implied that anti-cancer drugs would actually need to do the opposite — boost PKC activity. This study sets the stage for clinicians to one day use a pancreatic cancer patient’s PHLPP1/PKC levels as a predictor for prognosis, and for researchers to develop new therapeutic drugs that inhibit PHLPP1 and boost PKC as a means to treat the disease.

 

The ratio — high PHLPP1/low PKC — correlated with poor prognoses: no pancreatic patient with low PKC in the database survived longer than five-and-a-half years. On the flip side, 50 percent of the patients with low PHLPP1/high PKC survived longer than that. While still in the earliest stages, the researchers hope that this information might one day aid pancreatic diagnostics and treatment. The researchers are next planning to screen chemical compounds to find those that inhibit PHLPP1 and restore PKC levels in low-PKC-pancreatic cancer cells in the lab. These might form the basis of a new therapeutic drug for pancreatic cancer.

 

References:

 

https://health.ucsd.edu/news/releases/Pages/2019-03-20-two-enzymes-linked-to-pancreatic-cancer-survival.aspx?elqTrackId=b6864b278958402787f61dd7b7624666

 

https://www.ncbi.nlm.nih.gov/pubmed/30904392

 

https://www.ncbi.nlm.nih.gov/pubmed/29513138

 

https://www.ncbi.nlm.nih.gov/pubmed/18511290

 

https://www.ncbi.nlm.nih.gov/pubmed/28476658

 

https://www.ncbi.nlm.nih.gov/pubmed/28283201

 

https://www.ncbi.nlm.nih.gov/pubmed/24231509

 

https://www.ncbi.nlm.nih.gov/pubmed/28112438

 

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