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Saturday, April 4, 2020

Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells

You might just want this imaging to help you recover from a stroke and maybe prevent dementia. But that means your doctor and stroke hospital have to initiate contact with researchers to get human testing done. They don't contact researchers to get protocols for intervention so this will never occur. You're screwed.

Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells

Nature Communications volume 11, Article number: 1545 (2020) Cite this article

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Abstract

Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.

Introduction

The process of nuclear reprogramming to induced pluripotent stem cells (iPSCs) is characterized, upon completion, by a profound resetting of the epigenetic landscape of cells of origin, resulting in reversion of both cellular identity and age to an embryonic-like state^1,2,3,4.
Notably, if the expression of the reprogramming factors is only transiently applied and then stopped (before the so-called Point of No Return, PNR)⁵, the cells return to the initiating somatic cell state. These observations suggest that, if applied for a short enough time, the expression of reprogramming factors fails to erase the epigenetic signature defining cell identity; however, it remains unknown whether any substantial and measurable reprogramming of cellular age can be achieved before the PNR. First evidence that transient reprogramming can promote amelioration of aging phenotypes was shown by Ocampo et al., in progeroid mice carrying a Dox-inducible OSKM cassette⁶. Yet, important questions remain open. Murine genetic models of premature aging only in part recapitulate the complexity of natural aging, a phenomenon that is characterized by a slow and progressive accumulation of epigenetic errors. In addition, proof is lacking that the same rejuvenative effect can be achieved with naturally aged human cells isolated from elderly individuals, together with a comprehensive molecular and physiological analysis of the depth and extension of the rejuvenation in human cells. To address all these questions, we devised a platform that could let us test whether transient expression of nuclear reprogramming genes has any impact in ameliorating aging phenotypes in naturally aged human and mouse cells across multiple cell types and spanning all the hallmarks of aging.

Results

We first evaluated the effect of transient expression of reprogramming factors on the transcriptome of two distinct cell types—fibroblasts and endothelial cells—from aged human subjects, and we compared it with the transcriptome of the same cell types isolated from young donors (Fig. 1a, e). Fibroblasts were derived from arm and abdomen skin biopsies (25–35 years for the young control, n = 3, and 60–90 years for the aged group, n = 8), while endothelial cells were extracted from iliac vein and artery (15–25 years for the young control, n = 3, and 50–65 years for the aged group, n = 7). We utilized a non-integrative reprogramming protocol that we optimized, based on a cocktail of mRNAs expressing OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG (OSKMLN)⁷. Our protocol consistently produces iPSC colonies, regardless of age of the donors, after 12–15 daily transfections; we reasoned that the PNR in our platform occurs at about day 5 of reprogramming, based on the observation that the first detectable expression of endogenous pluripotency-associated lncRNAs occurs at day 5⁸. Therefore, we adopted a transient exogenous expression regimen where OSKMLN was daily transfected for 4 consecutive days, and performed gene expression analysis 2 days after the interruption (Fig. 1b).

**Fig. 1: Transcriptomic and epigenetic clock analysis shows more youthful signature upon transient expression of OSKMNL in human fibroblasts and endothelial cells.**

We performed paired-end bulk RNA sequencing on both cell types for the same three cohorts: young (Y), untreated aged (UA), and treated aged (TA). First, we compared the quantile normalized transcriptomes of young and untreated aged cells for each cell type (Y vs. UA) and found that 961 genes (5.85%) in fibroblasts (678 upregulated, 289 downregulated, Fig. 1a, c) and 748 genes (4.80%) in endothelial cells (389 upregulated, 377 downregulated, Fig. 1e, f) differed between young and aged cells, with the significance criteria of p < 0.05 and a log fold change cutoff ±0.5 (full list of genes in Supplementary Data 1 and 2). We found these sets of genes were enriched for many of the known aging pathways, identified in the hallmark gene set collection in the Molecular Signatures Database⁹ (Supplementary Data 3 and 4). When we mapped the directionality of expression above or below the mean of each gene, we could observe a clear similarity between treated and young cells as opposed to aged cells for both fibroblasts and endothelial cells (Fig. 1d, g). We further performed principal component analysis in this gene set space and determined that the young and aged populations were separable along the first principal component (PC1), which explained 64.8% of variance in fibroblasts and 60.9% of variance in endothelial cells. Intriguingly, the treated cells also clustered closer to the younger cells than the aged cells, simply along PC1 (Supplementary Fig. 1a, b).
Using the same significance criteria defined above, we then compared the treated and untreated aged populations (TA vs. UA) (Fig 1a, e, Supplementary Fig. 2 and Supplementary Data 5 and 6) and found that 1042 genes in fibroblasts (734 upregulated and 308 downregulated) and 992 in endothelial cells (461 upregulated and 531 downregulated) were differentially expressed. Interestingly, also within these sets of genes, we found enrichment for aging pathways, within the Molecular Signatures Database⁹ as previously described (Supplementary Data 7 and 8). When we compared the profiles young versus untreated aged (Y vs. UA) and untreated aged versus treated aged (UA vs. TA) in each cell type, we observed a 24.7% overlap for fibroblasts (odds ratio of 4.53, p < 0.05) and 16.7% overlap for endothelial cells (odds ratio of 3.84, p < 0.05) with the directionality of change in gene expression matching that of youth (i.e., if higher in young then higher in treated aged); less than 0.5% moved oppositely in either cell types (Supplementary Fig. 1a, b and Supplementary Data 9 and 10).
Next, we used these transcriptomic profiles to verify retention of cell identity after treatment. To this end, using established cell identity markers, we verified that none significantly changed upon treatment (Supplementary Data 11). In addition, we could not detect the expression of any pluripotency-associated markers (other than the OSKMLN mRNAs transfected in) (Supplementary Data 11). Altogether, the analysis of the transcriptomic signatures revealed that OSKLMN expression promotes a very rapid activation of a more youthful gene expression profile, which is cell-type specific, without affecting the expression of cell identity genes.
Epigenetic clocks based on DNA methylation levels are the most accurate molecular biomarkers of age across tissues and cell types and are predictive of a host of age-related conditions including lifespan^3,10,11,12. Exogenous expression of canonical reprogramming factors (OSKM) is known to revert the epigenetic age of primary cells to a prenatal state³. To test whether transient expression of OSKMLN could reverse the epigenetic clock of human somatic cells, we used two epigenetic clocks that apply to human fibroblasts and endothelial cells: Horvath’s original pan-tissue epigenetic clock (based on 353 cytosine–phosphate–guanine pairs), and the more recent skin-and-blood clock (based on 391 CpGs)^3,13.
According to the pan-tissue epigenetic clock, transient OSKMLN significantly (two-sided mixed-effect model P value = 0.023) reverted the DNA methylation age (average age difference = −3.40 years, standard error 1.17). The rejuvenation effect was more pronounced in endothelial cells (average age difference = −4.94 years, SE = 1.63, Fig. 1i) than in fibroblasts (average age difference = −1.84, SE = 1.46, Fig. 1h). Qualitatively similar, but less significant results could be obtained with the skin-and-blood epigenetic clock (overall rejuvenation effect −1.35 years, SE = 0.67, one-sided mixed-effect model P value = 0.042, and average rejuvenation in endothelial cells and fibroblasts is −1.62 years and −1.07, respectively).
Prompted by these results, we next analyzed the effect of transient reprogramming on various hallmarks of cellular physiological aging. We employed a panel of 11 established assays, spanning the hallmarks of aging¹⁴ (Supplementary Data 12), and performed most of the analyses using single-cell high-throughput imaging to capture quantitative changes in single cells and distribution shifts in the entire population of cells. All the analyses were performed separately in each individual cell line (total of 19 fibroblast lines: 3 young, 8 aged, and 8 treated aged; total 17 endothelial cell lines: 3 young, 7 aged, and 7 treated aged) (Fig. 2a and Supplementary Figs. 2–5). Statistical analysis was conducted by randomly sampling 100 cells per sample; the data was subsequently pooled by group category (see “Materials and Methods” for a detailed description of the Statistical methods that were used). Control experiments were performed by adopting the same transfection scheme using mRNA encoding for green fluorescent protein (GFP) (Supplementary Figs. 6 and 7).

**Fig. 2: Transient OSKMNL expression reverts aged physiology toward a more youthful state in human fibroblasts and endothelial cells.**

To extend our previous findings on epigenetics, we quantitatively measured by immunofluorescence (IF) the epigenetic repressive mark H3K9me3, the heterochromatin-associated protein HP1γ, and the nuclear lamina support protein LAP2α (Fig 2b–d). Aged fibroblasts and endothelial cells showed a decrease in the nuclear signal for all three markers compared with young cells, as previously reported²; treatment of aged cells resulted in an increase of these markers in both cell types. Next, we examined both pathways involved in proteolytic activity of the cells by measuring formation of autophagosomes, and chymotrypsin-like proteasomal activity, both reported to decrease with age^15,16. Treatment increased both to levels similar to or even higher than young cells, suggesting that early steps in reprogramming promote an active clearance of degraded biomolecules (Fig. 2e, f).
In terms of energy metabolism, aged cells display decreased mitochondrial activity, accumulation of reactive oxygen species (ROS), and deregulated nutrient sensing^2,16,17. We therefore tested the effects of treatment on aged cells by measuring mitochondria membrane potential, mitochondrial ROS, and levels of Sirtuin1 protein (SIRT1) in the cells. Transient reprogramming increased mitochondria membrane potential in both cell types (Fig. 2g), while it decreased mitochondrial ROS (Fig. 2h) and increased SIRT1 protein levels in fibroblasts, similar to young cells (Supplementary Fig. 8). Senescence-associated beta-galactosidase staining showed a significant reduction in the number of senescent cells in aged endothelial cells but not in fibroblasts (Supplementary Fig. 8). This decrease was accompanied by a decrease in pro-inflammatory senescence-associated secretory phenotype cytokines again in endothelial cells and not in fibroblasts (Fig. 2i and Supplementary Fig. 8)^16,18,19. Last, in neither cell type did telomere length, measured by quantitative fluorescence in situ hybridization^2,20, show significant extension with treatment (Supplementary Fig. 8), suggesting that the cells did not dedifferentiate into a stem-like state in which telomerase activity would be reactivated, and in agreement with previous reports where activation of TERT was observed at later stages of nuclear reprogramming²¹.
Next, we assessed the perdurance of these effects and found that most were significantly retained after 4 and 6 days from the interruption of reprogramming (Supplementary Figs. 9 and 10). We then examined how rapidly these physiological rejuvenative changes manifest by repeating the same sets of experiments in fibroblasts and endothelial cells that were transfected for just 2 consecutive days. Remarkably, we observed that most of the rejuvenative effects could already be seen after 2 days of treatment, although most were more moderate (Supplementary Figs. 11 and 12).
Collectively, this data demonstrates that transient expression of OSKMLN can induce a rapid, persistent amelioration, and reversal of cellular age in human somatic cells at the transcriptomic, epigenetic, and cellular levels. Importantly, these data demonstrate that the process of cellular rejuvenation is engaged very early, rapidly, and broadly in the reprogramming process. These epigenetic and transcriptional changes occur before any epigenetic reprogramming of cellular identity takes place, a novel finding in the field.
With these indications of a beneficial effect on cellular aging, we next investigated whether transient expression of OSKMNL could also reverse the inflammatory phenotypes associated with aging. After obtaining preliminary evidence of this reversal in endothelial cells (Fig. 2j), we extended our analysis to osteoarthritis, a disease strongly associated with aging and characterized by a pronounced inflammatory spectrum affecting the chondrocytes within the joint²². We thus isolated chondrocytes from cartilage of six 60–70-year-old patients undergoing total joint replacement surgery owing to their advanced-stage OA, and compared the results of treatment with chondrocytes isolated from three young individuals (Fig. 3a). Transient OSKMLN expression was performed for 2 or 3 days, and the analysis performed after 2 days from interruption of reprogramming, though the more consistent effect across patients was with longer treatment. Treatment showed a significant reduction in intracellular mRNA levels of RANKL and iNOS2, as well as in levels of inflammatory factors secreted by the cells (Fig. 3b–d). In addition, we observed increased cell proliferation (Fig. 3e), increased ATP production (Fig. 3f), and decreased oxidative stress as revealed by reduced mitochondrial ROS and elevated RNA levels of antioxidant SOD2 (Fig. 3g, h), a gene that has been shown to be downregulated in OA²³. Finally, when we checked for retention of cellular identity, we observed that the treatment did not affect the expression level of SOX9 (a transcription factor core to chondrocyte identity and function) and significantly increased the level of expression of COL2A1 (the primary collagen in articular cartilage) (qRT-PCR in Fig 3i, j), suggesting retention of chrondrogenic cell identity. Together, these results show that transient expression of OSKMLN can promote a partial reversal of gene expression and cellular physiology in aged OA chondrocytes toward a healthier, more youthful state, suggesting a potential new therapeutic strategy to ameliorate the OA disease process.

**Fig. 3: Transient OSKMNL expression mitigates inflammatory phenotypes in diseased chondrocytes.**

Stem cell loss of function and regenerative capacity represents another important hallmark of aging¹⁴. We thus wanted to assess the effect of transient reprogramming on the age-related changes in somatic stem cells that impair regeneration. First, we tested the effect of transient reprogramming on mouse-derived skeletal muscle stem cells (MuSCs). We treated MuSCs for 2 days while they were kept in a quiescent state using an artificial niche²⁴. We conducted initial experiments with young (3 month) and aged (20–24 months) murine MuSCs isolated by FACS (Fig. 4a). Treatment of aged MuSCs reduced both time of first division, approaching the faster activation kinetics of quiescent young MuSCs^25,26, and mitochondrial mass²⁷ (Supplementary Fig. 13a, b). Moreover, treatment partially rescued the reduced ability of single MuSCs to form colonies^25,28 (Supplementary Fig. 13c). We further cultured these cells and observed that treatment did not change expression of the myogenic marker MyoD but instead improved their capacity to differentiate into myotubes (Supplementary Fig. 14a–d), suggesting that transient reprogramming does not disrupt the myogenic fate but can enhance the myogenic potential.

**Fig. 4: Transient OSKMNL expression restores aged muscle stem cell potency.**

Next, we wanted to test MuSC function and potency to regenerate new tissue in vivo. To do this, we transduced young, aged, or transiently reprogrammed aged MuSCs with a lentivirus-expressing luciferase and GFP, and then transplanted the cells into injured tibialis anterior (TiA) muscles of immunocompromised mice. Longitudinal bioluminescence imaging (BLI) initially showed that muscles transplanted with treated aged MuSCs showed the highest signal (day 4, Fig 4b, c), but became comparable with muscles with young MuSCs by day 11 post transplantation; conversely muscle with untreated aged MuSCs showed lower signals at all time points post transplantation (Fig. 4b, c). IF analysis further revealed higher numbers of donor-derived (GFP⁺) myofibers in TiAs transplanted with treated compared with untreated aged MuSCs (Fig. 4d, e). Moreover, the GFP⁺ myofibers from treated aged cells exhibited increased cross-sectional areas when compared with their untreated counterparts, and in fact even larger than the young controls (Fig. 4f). Together, these results suggest improved tissue regenerative potential of transiently reprogrammed aged MuSCs. After 3 months, all mice were subjected to autopsy, and no neoplastic lesions or teratomas were discovered (Supplementary Table 1). To test potential long-term benefits of the treatment, we induced a second injury 60 days after cell transplantation, and again observed that TiA muscles transplanted with transiently reprogrammed aged MuSCs yielded higher BLI signals (Fig. 4g).
Sarcopenia is an age-related condition that is characterized by loss of muscle mass and force production^29,30. Similarly, in mice muscle functions show progressive degeneration with age^31,32. We wanted to test whether transient reprogramming of aged MuSCs would improve a cell-based treatment in restoring physiological functions of muscle of older mice. To test this, we first performed electrophysiology to measure tetanic force production in TiA muscles isolated from young (4 months) or aged (27 months) immunocompromised mice. We found that TiA muscles from aged mice have lower tetanic forces compared with young mice, suggesting an age-related loss of force production (Fig. 4h). Next, we isolated MuSCs from aged mice (20–24 months). After treating aged MuSCs, we transplanted them into cardiotoxin-injured TiA muscles of aged (20 months) immunocompromised mice. We waited 30 days to give enough time to the transplanted muscles to fully regenerate. We then performed electrophysiology to measure tetanic force production. Muscles transplanted with untreated aged MuSCs showed forces comparable with untransplanted muscles from aged control mice (Fig. 4h). Conversely, muscles that received treated aged MuSCs showed tetanic forces comparable with untransplanted muscles from young control mice (Fig. 4h and Supplementary Fig. 15a). These results suggest that transient reprogramming in combination with MuSC-based therapy can restore physiological function of aged muscles to that of youthful muscles.
Last, we wanted to translate these results to human MuSCs. We repeated the study, employing operative samples obtained from patients in different age ranges (10–80 years old), and transducing them with GFP- and luciferase-expressing lentiviral vectors (Fig. 4a). As in mice, transplanted, transiently reprogrammed, aged human MuSCs resulted in increased BLI signals compared with untreated MuSCs from the same individual, and comparable with those observed with young MuSCs (Fig. 4i and Supplementary Fig. 16a, b). Interestingly, the BLI signal ratio between contralateral muscles with treated and untreated MuSCs was higher in the older age group (60–80 years old) than in the younger age groups (10–30 or 30–55 years old), suggesting that ERA restores lost functions to younger levels in aged cells (Fig. 4j). Taken together, these results suggest that transient reprogramming partially restores the potency of aged MuSCs to a degree similar to that of young MuSCs, without compromising their fate, and thus has potential as a cell therapy in regenerative medicine.
Nuclear reprogramming to iPSCs is a multi-phased process comprising initiation, maturation, and stabilization³³. Upon completion of such a dynamic and complex epigenetic reprogramming, iPSCs are not only pluripotent but also youthful. While proof of principle that transient reprogramming can exert a systemic rejuvenation in a genetic model of aging (progeroid mice), the proof that a multispectral cellular rejuvenation could be achieved in a cell-autonomous fashion in human cells isolated from naturally aged individuals was missing. Here we demonstrate that a non-integrative, mRNAs-based platform of transient cellular reprogramming can very rapidly reverse a broad spectrum of aging hallmarks in the initiation phase, when epigenetic erasure of cell identity has not yet occurred. We show that the process of rejuvenation occurs in naturally aged human and mouse cells, with restoration of lost functionality in diseased cells and aged stem cells while preserving cellular identity. Future studies are required to elucidate the mechanism that drives the reversal of the aged phenotype during cellular reprogramming, uncoupling it from dedifferentiation process^34,35. Our results are novel and represent a significant step toward the goal of reversing cellular aging, and have potential therapeutic implications for aging and aging-related diseases.

Methods

Human fibroblast isolation and culture

Isolation was performed at Coriell Institute on healthy patients and from Alzheimer patient samples at Stanford Hospital, in accordance to the methods and protocols approved by the Institutional Review Board of Stanford University, biopsied for skin mesial aspect of mid-upper arm or abdomen using 2-mm punch biopsies from both male and female patients 60–70 years old (n = 8) and 25–35 years (n = 3). Cells were cultured out from these explants and maintained in Eagle’s Minimum Essential Medium with Earl’s salts supplemented with nonessential amino acids, 10% fetal bovine serum, and 1% Penicillin/Streptomycin. Cells were cultured at 37 °C with 5% CO₂.

Human endothelial cell isolation and culture

Isolation was performed at Coriell Institute from iliac arteries and veins, and muscle biopsies from Stanford Hospital, in accordance to the methods and protocols approved by the Institutional Review Board of Stanford University, from otherwise healthy 45–60 years old (n = 7). Tissue was digested with collagenase and cells released from the lumen were used to initiate cultures. Plates for seeding were coated with 2% gelatin, then washed with PBS before use. Cells were maintained in Medium 199 supplemented with 2 mM l-glutamine, 15% fetal bovine serum, 0.02 mg/ml Endothelial Growth Supplement, 0.05 mg/ml Heparin, and 1% Penicillin/Streptomycin. Cells were cultured at 37 °C with 5% CO₂.

Human articular chondrocyte isolation and culture

In accordance to the methods and protocols approved by Institutional Review Board of Stanford University, the human OA chondrocytes were derived from discarded tissues of OA patients (50–72 years of age, n = 6) undergoing total knee arthroplasty. The samples were surgical waste and were fully deidentified prior to procurement, hence no prior patient consent was required. Cartilage pieces were shaved off bone by scalpel, taking care to avoid any fat, then digested with collagenase in DMEM/F12 media (supplemented with 25 mg/ml ascorbate, 2 mM l-glutamine, 1% penicillin/streptomycin antibiotics, and 10% fetal bovine serum) for 1–2 days until shavings were substantially dissolved. Supernatant from cultures was strained, filtered, and centrifuged, and the cells were then resuspended in fresh media. The chondrocytes were cultured in high-density monolayer at 37 °C with 5% CO₂.

Mice

C57BL/6 male and NSG mice were obtained from Jackson Laboratory. NOD/MrkBomTac-Prkdcscid mice were obtained from Taconic Biosciences. Mice were housed and maintained in the Veterinary Medical Unit at the Veterans Affairs Palo Alto Health Care Systems. The Administrative Panel on Laboratory Animal Care of Stanford University approved animal protocols.

Human skeletal muscle specimens

The human muscle biopsy specimens were taken after patients (10–30 years, n = 2; 30–55 years, n = 2; 60–80 years, n = 3) gave informed consent as part of a human studies research protocol that was approved by the Stanford University Institutional Review Board. Sample processing for cell analysis began within 1–12 h of specimen isolation. In all studies, standard deviation reflects variability in data derived from studies using true biological replicates (i.e., unique donors). Data were not correlated with donor identity.

MuSC isolation and purification

Muscles were harvested from mouse hind limbs (n = 4) and mechanically dissociated to yield a fragmented muscle suspension. This was followed by a 45–50-min digestion in a Collagenase II-Ham’s F10 solution (500 U ml⁻¹, Invitrogen). After washing, a second digestion was performed for 30 min with Collagenase II (100 U ml⁻¹) and Dispase (2 U ml⁻¹, ThermoFisher). The resulting cell suspension was washed, filtered, and stained with VCAM-biotin, CD31-FITC, CD45-APC, and Sca-1-Pacific-Blue antibodies, all at dilutions of 1:100. Human MuSCs were purified from fresh operative samples. Operative samples were carefully dissected from adipose and fibrotic tissue and a dissociated muscle suspension prepared as described for mouse tissue. The resulting cell suspension was then washed, filtered, and stained with anti-CD31-Alexa Fluor 488, anti-CD45-Alexa Fluor 488, anti-CD34-FITC, anti-CD29-APC, and anti-NCAM-Biotin antibodies. Unconjugated primary antibodies were then washed and the cells were incubated for 15 min at 4 °C in streptavidin-PE/Cy7 to detect NCAM-biotin. Cell sorting was performed on calibrated BD-FACSAria II or BD FACSAria III flow cytometers equipped with 488-, 633-, and 405-nm lasers to obtain the MuSC population. A small fraction of sorted cells was plated and stained for Pax7 and MyoD to assess the purity of the sorted population.

mRNA transfection

Cells were transfected using either mRNA-In (mTI Global Stem) for fibroblasts and chondrocytes, to reduce cell toxicity, or Lipofectamine MessengerMax (ThermoFisher) for endothelial cells and MuSCs, which were more difficult to transfect, using the manufacturer’s protocol. For fibroblast and endothelial cells, serum free Pluriton medium with bFGF was used for transfection, while muscle stem cells and chondrocytes were kept in their original media—the former lacking serum and the later requiring serum to prevent the natural dedifferentiation of chondrocytes in culture. Culture medium was changed for fibroblasts and endothelial cells 4 h after transfection, but not for chondrocytes or MuSCs as overnight incubation was needed to produce a significant uptake of mRNA. Efficiency of delivery was confirmed by both GFP mRNA and immunostaining for individual factors in OSKMLN cocktail, the former also being used as a transfection control with the same protocol.

Immunocytochemistry

Cells were washed with HBSS/CA/MG and then fixed with 15% paraformaldehyde in PBS for 15 min. Cells were then blocked for 30 min to 1 h with a blocking solution of 1% BSA and 0.3% Triton X-100 in PBS for fibroblasts, endothelial cells, and 20% donkey serum/0.3% Triton in PBS for MuSCs. Primary antibodies were then applied in blocking solution and allowed to incubate overnight at 4 °C. The following day, the cells were washed with HBSS/CA/MG or PBST for MuSCs before switching to the corresponding Alexa Fluor-labeled secondary antibodies and incubated for 2 h. The cells were then washed again and stained with DAPI for 30 min and switched to HBSS/CA/MG for imaging or Fluoview for MuSCs.

Autophagosome formation staining

Cells were washed with HBSS/Ca/Mg and switched to a staining solution containing a proprietary fluorescent autophagosome marker (Sigma). The cells were then incubated at 37 °C in 5% CO₂ for 20 min, washed two times using HBSS/Ca/Mg, and stained for 15 min using CellTracker Deep Red cell labeling dye. Cells were then switched to HBSS/Ca/Mg for single-cell imaging using the Operetta High-Content Imaging System (Perkin Elmer).

Proteasome activity measurement

Wells were first stained with PrestoBlue Cell Viability dye (Life Technologies) for 10 min. Well signals were read using a TECAN fluorescent plate reader as a measure of cell count. Then cells were washed with HBSS/Ca/Mg before switching to original media containing the chymotrypsin-like fluorogenic substrate LLVY-R110 (Sigma), which is cleaved by proteasome 20S core particle. Cells were then incubated at 37 °C in 5% CO₂ for 2 h before signals were again read on the TECAN fluorescent plate reader. Readings were then normalized by PrestoBlue cell count.

Mitochondrial membrane potential staining

Tetramethylrhodamine Methyl Ester Perchlorate (Thermo) was added to cell culture media. This dye is sequestered by active mitochondria based on their membrane potential. Cells were incubated for 30 min at 37 °C in 5% CO₂ and washed two times with HBSS/Ca/Mg before staining for 15 min using CellTracker Deep Red. Finally, cells were imaged in fresh HBSS/Ca/Mg using the Operetta High-Content Imaging System (Perkin Elmer).

Mitochondrial ROS measurement

Cells were washed with HBSS/Ca/Mg and then switched to HBSS/Ca/Mg containing MitoSOX (Thermo), a live-cell-permeant fluorogenic dye that selectively targeted to mitochondria and fluoresces when oxidized by superoxide. Cells were incubated for 10 min at 37 °C in 5% CO₂. Cells were then washed twice with HBSS/Ca/Mg, and stained for 15 min using CellTracker Deep Red. Finally, cells were imaged in fresh HBSS/Ca/Mg using the Operetta High-Content Imaging System (Perkin Elmer).

SAβGal histochemistry

Cells were washed twice with PBS then fixed with 15% Paraformaldehyde in PBS for 6 min. Cells were rinsed three times with PBS before staining with X-gal chromogenic substrate, which is cleaved by endogenous Beta galactosidase. Plates were kept in the staining solution, Parafilmed, to prevent from drying out, and incubated overnight at 37 °C with ambient CO₂. The next day, cells were washed again with PBS before switching to a 70% glycerol solution for imaging under a Leica bright-field microscope.

Fixed and live-cell imaging

Samples were imaged using fluorescent microscopes—the Operetta High-Content Imaging System (Perkin Elmer) or the BZ-X700 (Keyence)—and either a 10× or 20× air objective. Harmony (Operetta) or Volocity (BZ-X700) imaging software was used to adjust excitation and emission filters and came with preprogrammed Alexa Fluor filter settings which were used whenever possible. All exposure times were optimized during the first round of imaging, and then kept constant through all subsequent imaging.

Image analysis

Columbus (Operetta) or Image J (BZ-X700) was used for image analysis. Columbus software was to identify single cells utilizing DAPI of CellTracker Re d to delineate nuclear and cell boundaries and calculate the signal statistics for each cell. Image J was used for muscle fibers to calculate the percentage of area composed of collagen by using the color threshold plug-in to create a mask of only the area positive for collagen. That area was then divided over the total area of the sample, which was found using the free draw tool. All other fiber analyses were performed using Volocity software and manually counting fibers using the free draw tool.

Statistics

Statistical analysis for physiological hallmarks of aging was done as described previously in Miller et al.². Briefly, 100 cells were randomly selected from each experimental group (data depicted in Supplementary Figs. 2–5), and they were then pooled in a unique population of 800 cells for aged fibroblasts (100 cells × 8 individuals for both aged and aged treated); 300 cells for young fibroblasts (100 cells × 3 individuals); 700 cells for aged endothelial cells (100 cells × 7 individuals for both aged and aged treated); 300 cells for young endothelial cells (100 cells × 3 individuals). Box distribution plots display the fluorescence intensity quantification of 100 cells from each patient. Distributions were compared by statistical analysis by using multiple-comparison ANOVA. Arbitrary units for frequency distributions of different cell types should not be compared because staining was performed at different times. Matlab 2017 (MathWorks) was used for data presentation and analysis.

Cytokine profiling

This work was performed together with the Human Immune Monitoring Center at Stanford University. Cell media was harvested and spun at 400 rcf for 10 min at room temperature. The supernatant was then snap frozen with liquid nitrogen until analysis. Analysis was done using the human 63-plex kit (eBiosciences/Affymetrix). Beads were added to a 96-well plate and washed in a Biotek ELx405 washer. Samples were added to the plate containing the mixed antibody-linked beads and incubated at room temperature for 1 h followed by overnight incubation at 4 °C with shaking. Cold and room temperature incubation steps were performed on an orbital shaker at 500–600 rpm. Following the overnight incubation, plates were washed in a Biotek ELx405 washer and then biotinylated detection antibody added for 75 min at room temperature with shaking. Plates were washed as above and streptavidin-PE was added. After incubation for 30 min at room temperature, wash was performed as above and reading buffer was added to the wells. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument with a lower bound of 50 beads per sample per cytokine. Custom assay Control beads by Radix Biosolutions were added to all wells.

Antibodies

The following antibodies were used in this study. The source of each antibody is indicated. Rabbit::H3K9me3 (Abcam #ab8898 1:4000), LAP2α (Abcam #ab5162 1:500), SIRT1 (Abcam #ab7343 1:200); Rabbit: Mouse: HP1γ (Millipore Sigma #05-690 1:200), Lamin A/C (Abcam #ab40567 1:200), GFP (Invitrogen, #A11122, 1:250); Luciferase (Sigma-Aldrich, #L0159, 1:200); Collagen I (Cedarlane Labs, #CL50151AP, 1:200); HSP47 (Abcam, #ab77609, 1:200), Laminin (Abcam, #AB11576, 1:1000), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2/16; BioLegend; #303008, 1:75) and anti-NCAM-Biotin (clone HCD56; BioLegend; #318319, 1:75), anti-CD31-Alexa Fluor 488 (clone WM59; BioLegend; #303110, 1:75), anti-CD45-Alexa Fluor 488 (clone HI30; Invitrogen; #MHCD4520, 1:75), anti-CD34-FITC (clone 581; BioLegend; #343503, 1:75), anti-CD29-APC (clone TS2/16; BioLegend; #303008, 1:75), and anti-NCAM-biotin (clone HCD56; BioLegend; #318319, 1:75).

RNA sequencing and data analysis

Cells were washed and digested by TRIzol (Thermo). Total RNA was isolated using the Total RNA Purification Kit (Norgen Biotek Corp) and RNA quality was assessed by the RNA analysis screentape (R6K screentape, Agilent); RNA with RIN > 9 was reverse transcribed to cDNA. cDNA libraries were prepared using 1 μg of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina). RNA quality was assessed by an Agilent Bioanalyzer 2100; RNA with RIN > 9 was reverse transcribed to cDNA. cDNA libraries were prepared using 500 ng of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina) with the added benefit of molecular indexing. Prior to any PCR amplification steps, all cDNA fragment ends were ligated at random to a pair of adapters containing a 8-bp unique molecular index. The molecular indexed cDNA libraries were than PCR amplified (15 cycles) and then QC’ed using a Bioanalzyer and Qubit. Upon successful QC, they were sequenced on an Illumina Nextseq platform to obtain 80-bp single-end reads. The reads were trimmed by 2 nt on each end to remove low-quality parts and improve mapping to the genome. The 78-nt reads that resulted were compressed by removing duplicates, while keeping track of how many times each sequence occurred in each sample in a database. The unique reads were then mapped to the human genome using exact matches. This misses reads that cross exon–exon boundaries, as well as reads with errors and SNPs/mutations, but it does not have substantial impact on estimating the levels of expression of each gene. Each mapped read was then assigned annotations from the underlying genome. In case of multiple annotations (e.g., a miRNA occurring in the intron of a gene), a hierarchy based on heuristics was used to give a unique identity to each read. This was then used to identify the reads belonging to each transcript and coverage over each position on the transcript was established. This coverage is nonuniform and spiky. Therefore, we used the median of this coverage as an estimate of the expression value of each gene. In order to compare the expression levels in different samples, quantile normalization was used. Further data analysis was done in Matlab. Ratios of expression levels were then calculated to estimate the log (base 2) of the fold change. Student’s t test was used to determine significance with a p < 0.05 cutoff. Molecular Signatures Database categorization was done using Broad Institute online tools https://software.broadinstitute.org/gsea/msigdb/.

Gene expression analysis

Total RNA was purified using the RNeasy Plus Mini kit (Qiagen), and cDNA was prepared with the First-strand cDNA synthesis kit (Applied Biosystems). The quantitative polymerase chain reaction was performed using VeriQuest Mastermix (ThermoFisher Scientific) for SYBR Green and Taqman primer sets, respectively. The relative gene expression was analyzed by the ΔΔCt method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The Taqman probes for human GAPDH (Hs02758991); COL2A1 (Hs00264051); SOX9 (Hs00165814); MMP3 (Hs00233962) and MMP13 (Hs00233992) were purchased from Applied Biosystems. The SYBR green primer sequences used are: Human SOD2 (F)-5′GGC CTA CGT GAA CAA CCT GA3′; Human SOD2 (R)-5′TGG GCT GTA ACA TCT CCC TTG3′; Human iNOS (F)-5′GTC CCG AAG TTC TCA AGG CA3′; Human iNOS (R)-5′GTT CTT CAC TGT GGG GCT TG3′; Human RANKL (F)-5′CAG GTT GTC TGC AGC GT3′ and Human RANKL (R)-5′GAT CCA TCT GCG CTC TGA AAT A3′; Human GAPDH (F)- 5′TGT CCC CAC TGC CAA CGT GTC3′; Human GAPDH (R)-5′AGC GTC AAA GGT GGA GGA GTG GGT3′.

ATP assay

ATP in the chondrocytes was measured using colorimetric assay and the ATP assay kit (ab83355; Abcam, Cambridge, MA) following the manufacturer’s instructions. Cells were washed in cold phosphate buffered saline and homogenized and centrifuged to collect the supernatant. The samples were loaded with assay buffer in triplicate. ATP reaction mix and background control (50 µL) was added to the wells and incubated for 30 min in dark. The plate was read at OD 570 nm using SpectraMax M2e (Molecular Devices, Sunnyvale, CA). The mean optical density was used to estimate of the intracellular ATP concentration relative to the standard curve.

Cell proliferation assay

Cell viability was assayed using the PrestoBlue Cell Viability (Life Technologies) reagent consecutively for 3 days post transfection in accordance with the manufacturer’s instructions. PrestoBlue reagent was added to the cell culture medium, and the cells were incubated at 30 °C for 30 min. Absorbance of the PrestoBlue was measured daily using SpectraMax M2e (Molecular Devices, Sunnyvale, CA).

EDU staining

Staining was done according to the manufacturer’s protocol using the Click-iT EdU kit. Cells were labeled with Edu after switching to growth media. Cells were allowed to grow 1 or 2 days before fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton X-100 in PBST. Cells were the incubated in Click-It reaction cocktail for 30 min before washing in PBS and imaging.

MitoTracker staining and flow cytometry analysis

Cells were washed twice with Ham’s F10 (no serum or pen/strep). Subsequently, MuSCs were stained with MitoTracker Green FM (ThermoFisher, M7514) and DAPI for 30 min at 37 °C, washed three times with Ham’s F10, and analyzed using a BD FACSAria III flow cytometer.

Myogenic colony-forming cell assay for MuSCs

Single treated and control MuSCs were deposited into wells of collagen- and laminin-coated plates at one cell per well by BD FACSAria III flow cytometer. Collagen/laminin coating was accomplished by overnight incubation of the plates rocking at 4 °C with a 1:1 mixture of laminin (10 μg/ml ThermoFisher 23017-015) and collagen (10 μg/ml Sigma C8919) in PBS. Coated wells were washed three times with PBS before use. The cells were cultured in grow media, F10 medium supplemented with 20% horse serum, and 5 ng/ml basic fibroblast growth factor (bFGF; PeproTech 100-18B). After 6 days of culture, plates were fixed with 4% paraformaldehyde (Electron Microscopy Services 15710), stained with DAPI (Invitrogen D1306), and scored by microscopy to determine the number of myogenic colony-forming cells, defined by wells that contained at least eight cells.

Myogenic/fusion index

Myogenic analysis was completed as previously described³⁶. After MuSCs underwent reprogramming or control treatment, cells were cultured in grow media. To induce differentiation, myoblast cultures were maintained in DMEM supplemented with 2% horse serum. The myogenic/fusion index was determined as the percentage of myonuclei in myotubes (defined as cells with three or more nuclei) compared with the total number of nuclei in the field.

Lentiviral transduction

Luciferase and GFP protein reporters were subcloned into a third-generation HIV-1 lentiviral vector (CD51X DPS, SystemBio). To transduce freshly isolated MuSCs, cells were plated on a Poly-D-Lysine (Millipore Sigma, A-003-E) and ECM coated eight-well chamber slide (Millipore Sigma, PEZGS0896) and were incubated with 5 μl of concentrated virus per well and 8 μg/mL polybrene (Santa Cruz Biotechnology, sc-134220). Plates were spun for 5 min at 3200 g, and for 1 h at 2500 g at 25 °C. Cells were then washed with fresh media two times, scraped from plates, and resuspended in the final volume according to the experimental conditions.

Bioluminescence imaging

Bioluminescent imaging was performed using the Xenogen IVIS-Spectrum System (Caliper Life Sciences). Mice were anesthetized using 2% isoflurane at a flow rate of 2.5 l/min Intraperitoneal injection of d-Luciferin (50 mg/ml, Biosynth International Inc.) dissolved in sterile PBS was administered. Immediately following the injection, mice were imaged for 30 s at maximum sensitivity (f-stop 1) at the highest resolution (small binning). Every minute, a 30-s exposure was taken until the peak intensity of the bioluminescent signal began to diminish. Each image was saved for subsequent analysis.

Bioluminescence image analysis

Analysis of each image was performed using Living Image Software, version 4.0 (Caliper Life Sciences). A manually generated circle was placed on top of the region of interest and resized to completely surround the limb or the specified region on the recipient mouse. Similarly, a background region of interest was placed on a region of a mouse outside the transplanted leg.

Tissue harvesting

TiA muscles were carefully dissected away from the bone, weighed, and placed into a 0.5% PFA solution for fixation overnight. The muscles were then moved to a 20% sucrose solution for 3 h or until muscles reached their saturation point and began to sink. The tissues were then embedded and frozen in Optimal Cutting Temperature (OCT) medium and stored at −80 °C until sectioning. Sectioning was performed on a Leica CM3050S cryostat that was set to generate 10-μm sections. Sections were mounted on Fisherbrand Colorfrost slides. These slides were stored at −20 °C until immunohistochemistry could be performed.

Flow cytometry

For mouse MuSC sorting scheme, we followed the same gating strategy previously published³⁷. For human MuSC sorting scheme, we followed the same strategy previously published³⁸.

Histology

TiA muscles were fixed for 5 h using 0.5% electron-microscopy-grade paraformaldehyde and subsequently transferred to 20% sucrose overnight. Muscles were then frozen in OCT, cryosectioned at a thickness of 10 μm, and stained. For colorimetric staining with Hematoxylin and Eosin (Sigma) or Gomori Trichrome (Richard-Allan Scientific), samples were processed according to the manufacturer’s recommended protocols.

Ex vivo force measurement

To measure the force, we isolated the TiA in a bath of oxygenated Ringer’s solution and stimulated it with plate electrodes. Immediately after euthanasia, the distal tendon of the TiA, the TiA, and the knee (proximal tibia, distal femur, patella, and associated soft tissues) were dissected out and placed in Ringer’s solution (Sigma) maintained at 25 °C with bubbling oxygen with 5% carbon dioxide. The proximal tibia was sutured to a rigid wire attached to the force transducer, and the distal tendon was sutured to a rigid fixture. No suture loops or slack was present in the system. The contralateral limb was immediately dissected and kept under low passive tension in oxygenated Ringer’s solution bath until measurement. Supramaximal stimulation voltage was found, and the active force-length curve was measured in a manner similar to the in vivo condition. After measurement, the muscle was dissected free and the mass measured. An Aurora Scientific 1300-A Whole Mouse Test System was used to gather force production data.

DNA methylation data

The human Illumina Infinium EPIC 850K chip was applied to n = 16 DNA samples (corresponding to two treatment levels (before/after treatment) of four fibroblasts and four endothelial cells). The raw image data were normalized using the “preprocessQuantile” normalization method implemented in the “minfi” R package^39,40.

Epigenetic clock analysis

Several DNAm-based biomarkers have been proposed in the literature, which differ in terms of their applicability (most were developed from blood), and in terms of their biological interpretation (reviewed in ref. ¹¹). We focused on two epigenetic clocks that apply to fibroblasts and endothelial cells. In our primarily analysis, we used the pan-tissue epigenetic clock³ because it applies to all sources of DNA (with the exception of sperm). A previously defined mathematical algorithm is used to combine the methylation levels of 353 CpG into an age estimate (in units of years), which is referred to as epigenetic age or DNAm age³. In our secondary analysis, we used the skin-and-blood epigenetic clock (based on 391 CpGs) because it is known to lead to more accurate DNAm age estimates in fibroblasts, keratinocytes, buccal cells, blood cells, saliva, and endothelial cells¹³.
We used the online version of the epigenetic clock software to arrive at DNA methylation age estimates from n = 16 samples collected from n = 8 individuals³. Although the chronological age range was relatively narrow (ranging from 47 to 69 years, median age = 55), the two DNAm age estimates exhibited moderately high correlations with chronological age (r = 0.42 and r = 0.63, P = 0.0089 for the pan-tissue- and the skin-and-blood clock, respectively).
Two samples (before and after rejuvenation treatment) were generated from each of n = 8 individuals. To properly account for the dependence structure in the data, we used linear mixed effects models to regress DNAm age (dependent variable) on treatment status, chronological age, and individual identifier (coded as random effect). Toward this end, we used the “lmer” function in the “lmerTest” R package⁴¹.

Reporting summary

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

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The data used for the methylation clock analysis will be available through the following GSE number starting April 08 2020: GSE142439. RNASeq data have been deposited to the Sequence read Archive (SRA), and will be available upon publication through the following SRA number: PRJNA598923.

One handed bookshelf use

This is nigh onto impossible one handed, getting a book into a pretty full shelf doesn't work one-handed. No training in this ADL.

This one requires me to use my elbow to hold the left side as I push them together.


My travel books, because the left side goes against the lip I can actually get books into here and push the holders closed.

Friday, April 3, 2020

Chilling concussed cells shows promise for full recovery

All my previous research posts on this for stroke suggested no useful intervention. Obviously no protocols(either positive or negative) were ever written on hypothermia so everyone is still flailing in the dark. The result being that survivors are still screwed with no consequences to the doctors/researchers who haven't written up protocols on this. Don't you just love incompetence?

hypothermia (46 posts)

Chilling concussed cells shows promise for full recovery

University of Wisconsin-Madison

MADISON, Wis. -- In the future, treating a concussion could be as simple as cooling the brain.
That's according to research conducted by University of Wisconsin-Madison engineers, whose findings support the treatment approach at the cellular level.
"There are currently no effective medical treatments for concussions and other types of traumatic brain injuries," says Christian Franck, the UW-Madison associate professor of mechanical engineering who led the study. "We're very excited about our findings because they could potentially pave the way for treatments we can offer patients."
The process is a bit more finicky than just applying an ice pack to the head.
Conducting experiments on brain cells in a dish, Franck and his team discovered several key parameters that determined the effectiveness of therapeutic cooling for mitigating damage to the injured cells.
"We found that, for this treatment to be successful, there's a sweet spot," he says. "You can't cool too little; you can't cool too much; and you can't wait too long following an injury to start treatment."
And when the researchers identified that sweet spot, the results were striking.
"I was amazed at how well the cooling worked," Franck says. "We actually went back and repeated the experiments multiple times because I didn't believe it at first."
The researchers published their findings in the journal PLOS ONE.
The high occurrence of concussions underscores the pressing need for treatments. Every year in the United States, there are an estimated 1.7 million new cases of traumatic brain injury assessed in emergency rooms, and the incidence of sports-related concussions may approach 3.8 million annually.
A traumatic impact to the brain can turn on biochemical pathways that lead to neurodegeneration, the progressive deterioration and loss of function in brain cells. Neurodegeneration causes long-lasting and potentially devastating health issues for patients.
"These pathways are like flipping on a bad molecular switch in your brain," says Franck.
In their experiments, the researchers looked at two of those biochemical pathways.
First, they created a network of neurons in a dish and delivered a mechanical stimulus that simulates the kind of injury and cell damage that people experience with a concussion.
Then they cooled the injured cells separately to four different temperatures. They found that 33 degrees Celsius (91.4 degrees Fahrenheit) provided the most protective benefit for the cells after 24 and 48 hours post-injury. Notably, cooling to 31 degrees Celsius had a detrimental effect.
"So there's such a thing as cooling too much," Franck says.
Time also is a factor. For the best outcome, the team determined that cooling needed to begin within four hours of the injury and continue for at least six hours, although Franck says cooling for even 30 minutes still showed some benefits.
When they adhered to those parameters, the researchers discovered they could keep the cells' damaging biochemical pathways switched off. In other words, the cells remained healthy and functioning normally--even though they had just suffered a traumatic injury.
After six hours of cooling, the researchers brought the concussed brain cells back up to normal body temperature, curious about whether warming would cause the damaging biochemical pathways to turn on.
"The biggest surprise was that the molecular switches actually stayed off -- permanently -- through the duration of the lab experiment," Franck says. "That was huge."
He and his students compared their results with previous animal studies and randomized human trials that investigated cooling as a treatment for traumatic brain injuries.
"We found really good agreement between the studies when we dialed in to those specific parameters, so that's a very encouraging sign," Franck says. "But this isn't the end of the story. We think this warrants further investigation in animal studies."
Franck says there's more to learn before cooling the brain could be a practical treatment for patients at a clinic. For example, it's not as easy as simply lowering the temperature of a person's whole body, which taxes the heart and can have a strong negative effect on the immune system.
Rather, isolating cooling to the brain is crucial. "We hope our paper will spawn renewed motivation and interest in solving the technical challenges for getting this type of treatment to patients in the future," Franck says. "For a long time, the scientific literature was inconclusive on whether this would be a successful treatment. What we showed in our study was that, yes, as far as the cell biology is concerned, this is effective. And so now it's really worth thinking about how we might implement this in practice."

###

Researchers from Brown University collaborated on this research through the U.S. Office of Naval Research-sponsored PANTHER interdisciplinary research program at UW-Madison for studying traumatic brain injuries. Franck is the Grainger Institute for Engineering Associate Professor and PANTHER director.
DOWNLOAD IMAGES: https://uwmadison.box.com/v/cooling-concussions
--Adam Malecek, acmalecek@wisc.edu

Higher vitamin B12 level at Parkinson's disease diagnosis is associated with lower risk of future dementia

With your risk of getting Parkinsons as a result of your stroke, JUST MAYBE YOU WANT YOUR DOCTOR TO BE COMPETENT ENOUGH TO GIVE YOU THE CORRECT PROTOCOL ON B12. UP TO YOU HOW YOU WANT TO HANDLE THIS, LET IT SLIDE OR CONTACT THE BOARD OF DIRECTORS AND HAVE EVERYONE FIRED.

My Dad had Parkinson's with dementia, I am not going down that route.

Look at the dosing info here from WebMD. But remember you can do nothing without your doctor's prescription.

VITAMIN B12

Parkinson’s Disease May Have Link to Stroke March 2017

Higher vitamin B12 level at Parkinson's disease diagnosis is associated with lower risk of future dementia

Published:March 17, 2020DOI:https://doi.org/10.1016/j.parkreldis.2020.03.009

Highlights

•
Treatment for non-motor features of Parkinson's disease are limited.
•
Higher risk of future dementia in patients with lower vitamin B12 levels at Parkinson's disease diagnosis.
•
Duration of Parkinson's disease symptoms and older age increase dementia risk.
•
Cognitively impaired Parkinson's disease patients tend to have lower B12 levels.
•
Vitamin B12 deserves further study for possible disease modifying effects in Parkinson's disease.

Abstract

Introduction

To determine whether vitamin B12 level at Parkinson's disease (PD) diagnosis predicts time to develop dementia.

Methods

We utilized a population-based cohort of Parkinsonism patients to examine the relationship between serum vitamin B12 at the time of PD diagnosis and dementia risk. Receiver operating curves were calculated for vitamin B12 cutoffs maximizing sensitivity and specificity for determining who developed dementia. Time from Parkinsonism diagnosis to dementia, death, or censoring was calculated utilizing Kaplan-Meier analysis and Cox-proportional hazard models.

Results

PD patients who did not develop dementia had higher baseline levels of vitamin B12 at PD diagnosis (648.5 ng/L vs 452 ng/L, p < 0.05) than those who developed dementia. Dementia risk was significantly lower in the 3rd tertile compared with 2nd tertile and trended towards significance compared to the 1st tertile. Each 100 unit increase in vitamin B12 level had a hazard ratio of 0.31 (95% CI 0.44–0.95) for future dementia (p < 0.05). Vitamin B12 cutoff of <587 ng/L was 87% sensitive and 70% specific (AUC 0.79, 95% CI 0.60–0.98) distinguishing patients with dementia. PD patients with vitamin B12 levels <587 ng/L were 5.4 times more likely to develop dementia, with 50% having dementia within 5 years of PD diagnosis compared with 11% in those with a vitamin B12 level of ≥587 ng/L (p < 0.05). Now we just need to know EXACTLY the daily dose needed per bodyweight.

Conclusion

Higher levels of serum vitamin B12 at PD diagnosis correlate with lower risk of future dementia. The role of vitamin B12 in the development of dementia among PD patients deserves further evaluation.

Highlights • Treatment for non-motor features of Parkinson's disease are limited. • Higher risk of future dementia in patients with lower vitamin B12 levels at Parkinson's disease diagnosis. • Duration of Parkinson's disease symptoms and older age increase dementia risk. • Cognitively impaired Parkinson's disease patients tend to have lower B12 levels. • Vitamin B12 deserves further study for possible disease modifying effects in Parkinson's disease. Abstract Introduction To determine whether vitamin B12 level at Parkinson's disease (PD) diagnosis predicts time to develop dementia. Methods We utilized a population-based cohort of Parkinsonism patients to examine the relationship between serum vitamin B12 at the time of PD diagnosis and dementia risk. Receiver operating curves were calculated for vitamin B12 cutoffs maximizing sensitivity and specificity for determining who developed dementia. Time from Parkinsonism diagnosis to dementia, death, or censoring was calculated utilizing Kaplan-Meier analysis and Cox-proportional hazard models. Results PD patients who did not develop dementia had higher baseline levels of vitamin B12 at PD diagnosis (648.5 ng/L vs 452 ng/L, p < 0.05) than those who developed dementia. Dementia risk was significantly lower in the 3rd tertile compared with 2nd tertile and trended towards significance compared to the 1st tertile. Each 100 unit increase in vitamin B12 level had a hazard ratio of 0.31 (95% CI 0.44–0.95) for future dementia (p < 0.05). Vitamin B12 cutoff of

Interim guidance issued on stroke care during COVID-19 pandemic

Notice the word 'care' NOT RECOVERY. The tyranny of low expectations on full display there. A lot of dead wood in stroke needs to be fired.

Interim guidance issued on stroke care during COVID-19 pandemic

DALLAS, April 2, 2020 — The American Heart Association/American Stroke Association’s Stroke Council Leadership has released “Temporary Emergency Guidance to U.S. Stroke Centers During the COVID-19 Pandemic,” which was published late yesterday in Stroke, a journal of the American Stroke Association, a division of the American Heart Association.
In broad terms, the Stroke Council Leadership recommends that U.S. stroke centers:

Adhere to treatment guidelines for patients to ensure appropriate stroke care is provided to the extent possible during the crisis;
seek ways to minimize the use of scarce personal protective equipment (PPE) and reduce the number of team members responding to emergency stroke patients;
increase the use of interactive videoconferencing in the remote delivery of acute stroke care, also known as telestroke;
follow their local health department, Centers for Disease Control and World Health Organization guidelines on hand washing, use of PPE and COVID-19 testing and evaluation;
continue to deliver multidisciplinary, collaborative stroke care to patients for a unified Stroke System of Care; and
ask medical personnel who are exposed or contract COVID-19 to self-quarantine as appropriate.

The Stroke Council Leadership noted, “While these recommendations have not yet undergone the traditional rigorous process of development, refinement and peer review … we acknowledge the mounting concern regarding optimal stroke care during the COVID-19 pandemic among vascular neurologists and those clinicians who care for patients with stroke. We issue this temporary statement as an interim stopgap opinion, pending a more thorough and considered process.”
The Stroke Council Leadership will continue to collect individual protocols and best practices and to evaluate and update the statement continuously during the crisis.
Additional Resources:

Available multimedia is on right column of release link https://newsroom.heart.org/news/interim-guidance-issued-on-stroke-care-during-covid-19-pandemic
Coronavirus – precautions and information for all
COVID-19 Content: An AHA Compendium – resources for health care professionals
Additional editorials on stroke care during COVID-19, recently published in Stroke
Follow AHA/ASA news on Twitter @HeartNews
Follow news from Stroke, the ASA/AHA journal @StrokeAHA_ASA

The Association receives funding primarily from individuals. Foundations and corporations (including pharmaceutical, device manufacturers and other companies) also make donations and fund specific Association programs and events. The Association has strict policies to prevent these relationships from influencing the science content. Revenues from pharmaceutical and device corporations and health insurance providers are available at https://www.heart.org/en/about-us/aha-financial-information.
American Stroke Association
The American Stroke Association is devoted to saving people from stroke(Nothing about getting them recovered.) — the No. 2 cause of death in the world and a leading cause of serious disability. We team with millions of volunteers to fund innovative research, fight for stronger public health policies and provide lifesaving tools and information to prevent and treat stroke(Absolutely NOTHING on your site about getting 100% recovered). The Dallas-based association officially launched in 1998 as a division of the American Heart Association. To learn more or to get involved, call 1-888-4STROKE or visit stroke.org. Follow us on Facebook and Twitter.

###

For Media Inquiries and AHA/ASA Expert Perspective: 214-706-1173
Michelle Kirkwood: 703-457-7838; michelle.kirkwood@heart.org
For Public Inquiries: 1-800-AHA-USA1 (242-8721)
heart.org and stroke.org

Thursday, April 2, 2020

Gait outcomes after additional backward walking training in patients with stroke: a randomized controlled trial

If your doctor didn't do anything with this from 15 years ago or any of the following then s/he needs to be fired. Why is your board of directors being so incompetent in not demanding new interventions from any stroke research?

Effectiveness of backward walking treadmill training in lower extremity function after stroke - Oct. 2006

“A Backward Walking Training Program to Improve Balance and Mobility in Acute Stroke: A Pilot Randomized Controlled Trial” Oct. 2017

Walking backwards boosts creativity April 2015

Gait outcomes after additional backward walking training in patients with stroke: a randomized controlled trial

Yea-Ru Yang
Institute & Faculty of Physical Therapy, National Yang-Ming University, Shih-Pai and
Jyh-Geng Yen
TaipeiMunicipal WanFang Hospital, Mu-Zha,
Ray-Yau Wang
Institute & Faculty of Physical Therapy, National Yang-Ming University,Shih-Pai,
Lu-Lu Yen
and
Fu-Kong Lieu
Department of Physical Medicine & Rehabilitation, Cheng Hsin Rehabilitation Medical Center, Shih-Pai, Taipei, Taiwan Received 23rd July 2004; returned for revisions 9th September 2004; revised manuscript accepted 13th October 2004.
Objective
: To examine the effectiveness of additional backward walking training on gait outcome of patients post stroke.
Design
: Randomized controlled trial.
Setting
: Medical centre.
Subjects
: Twenty-five subjects with stroke, who were lower extremity Brunnstrom motor recovery stage at 3 or 4 and were able to walk 11 m with or without a walking aid or orthosis, randomly allocated to two groups, control (n=12) and experimental (n=13).
Interventions
: Subjects in both groups participated in 40 min of conventional training programme three times a week for three weeks. Subjects in experimental group received additional 30 min of backward walking training for three weeks at a frequency of three times per week.
Main measures
: Gait was measured using the Stride Analyzer. Gait parameters of interest were walking speed, cadence, stride length, gait cycle and symmetry index. Measures were made at baseline before commencement of training(pretraining) and at the end of the three-week training period (post-training).
Results
: After a three-week training period, subjects in experimental group showed more improvement than those in control group for walking speed (change score: 8.60 ± 6.95 versus 3.65 ± 2.92, p
-value=0.032), stride length (change score:0.090 ± 0.076 versus 0.0064 ± 0.078, p
-value=0.006), and symmetry index(change score: 44.07 ± 53.29 versus 5.30 ± 13.91, p
-value=0.018).
Conclusions
: This study demonstrated that asymmetric gait pattern in patients post stroke could be improved from receiving additional backward walking therapy.
Address for correspondence: Yea-Ru Yang, Institute &Faculty of Physical Therapy, National Yang-Ming University,155, Sec 2, Li Nong St., Shih-Pai, Taipei, Taiwan.e-mail: yryang@ym.edu.tw
Clinical Rehabilitation

For stroke survivors, light physical activity linked to better daily function

Is this your hospitals 'Get out of jail free' card? Allowing them to declare rehab a success if you can do light physical activity? I would be screaming at my providers if this crapola is the best they are trying for. You need to demand 100% recovery.

For stroke survivors, light physical activity linked to better daily function

Apr 2, 2020 8:30 am by Diana Yates | Life Sciences Editor, U. of I. News Bureau | 217-333-5802Life Sciences

Kinesiology and community health professor Neha Gothe and her colleagues examined the relationship between physical activity and physical function in stroke survivors. They found that those who engaged in more light physical activity also reported fewer functional limitations.

Photo courtesy Neha Gothe

CHAMPAIGN, Ill. — Researchers used accelerometers to measure daily physical activity in 30 stroke survivors for a week, assessing how much the participants moved and how well they performed routine physical tasks. The study revealed that stroke survivors who engaged in a lot of light physical activity – taking leisurely walks or attending to nonstrenuous household chores, for example – also reported fewer physical limitations than their more sedentary peers.
The researchers describe their findings in the American Journal of Physical Medicine and Rehabilitation.
“Stroke is a major cause of disability in older adults,” said Neha Gothe, a professor of kinesiology and community health at the University of Illinois at Urbana-Champaign who led the research. “We know that physical activity can improve how well people survive a stroke and recover after the fact. But almost no research has looked at how physical activity of different intensities affects physical function among stroke survivors.”
Gothe and her research team used two measures of physical ability – the Short Physical Performance Battery, which measures balance, walking speed and lower-limb endurance, and the Late-Life Function and Disability Instrument, which asks participants to report how difficult it is for them to perform daily tasks such as getting in and out of a car or pouring water from a heavy pitcher.
The researchers discovered that, on average, the stroke survivors logged only about seven minutes of moderate-to-vigorous activity per day.
“In contrast, they averaged more than three hours of light physical activity each day,” Gothe said. “This includes things like walking at a leisurely pace, housekeeping, light gardening or other activities that do not cause a person to break a sweat.”
The amount of moderate-to-vigorous physical activity was the best predictor of participants’ performance on objective measures of physical function, the researchers found. But a person’s self-reported ability to perform daily tasks was much more closely associated with the amount of time they engaged in light physical activity.
“Our findings are preliminary but suggest that – in addition to moderate-to-vigorous physical activity – those daily routines that keep us on our feet and physically engaged in lighter tasks also contribute to better physical functioning in stroke survivors. This aligns with the latest Physical Activity Guidelines for Americans that emphasize the need to move more and sit less,” Gothe said. “Engaging in light physical activity can be healthy and beneficial, especially for those with chronic health conditions such as stroke.”

Editor’s notes:
To reach Neha Gothe, email npg@illinois.edu.
The paper “Associations between physical activity intensities and physical function in stroke survivors” is available online and from the U. of I. News Bureau.
DOI: 10.1097/PHM.0000000000001410

Contemporary Bobath Part 3: Scoping review of Bobath studies

I'm not going to read it but just in case your therapist still uses it even after it was proven not to work since 2003. You'll have to ask your therapist why it is still in use. And ask your stroke hospital why the fuck they are still allowing therapy that doesn't work. People need to be fired, starting with the board of directors. Unless you prefer incompetent hospitals.

Physiotherapy Based on the Bobath Concept for Adults with Post-Stroke Hemiplegia: A Review of Effectiveness Studies 2003

The latest here:

Contemporary Bobath Part 3: Scoping review of Bobath studies

State of the Art and Future Directions for Lower Limb Robotic Exoskeletons

3 years old. Your doctor is responsible for KNOWING AND USING THE STATE OF ART TODAY!

State of the Art and Future Directions for Lower Limb Robotic Exoskeletons

Some Coronavirus Patients Show Signs of Brain Ailments

Be careful out there.

Some Coronavirus Patients Show Signs of Brain Ailments

Doctors have observed neurological symptoms, including confusion, stroke and seizures, in a small subset of Covid-19 patient.

A coronavirus patient being transferred from a full hospital in Brussels, Belgium, on Wednesday. — Credit...Francisco Seco/Associated Press

By Roni Caryn Rabin

April 1, 2020

Neurologists around the world say that a small subset of patients with Covid-19 are developing serious impairments of the brain.

Although fever, cough and difficulty breathing are the typical hallmarks of infection with the new coronavirus, some patients exhibit altered mental status, or encephalopathy, a catchall term for brain disease or dysfunction that can have many underlying causes, as well as other serious conditions. These neurological syndromes join other unusual symptoms, such as diminished sense of smell and taste as well as heart ailments.

In early March, a 74-year-old man came to the emergency room in Boca Raton, Fla., with a cough and a fever, but an X-ray ruled out pneumonia and he was sent home. The next day, when his fever spiked, family members brought him back. He was short of breath, and could not tell doctors his name or explain what was wrong — he had lost the ability to speak.

The patient, who had chronic lung disease and Parkinson’s, was flailing his arms and legs in jerky movements, and appeared to be having a seizure. Doctors suspected he had Covid-19, and were eventually proven right when he was finally tested.

Continue reading the main story

On Tuesday, doctors in Detroit reported another disturbing case involving a female airline worker in her late 50s with Covid-19. She was confused, and complained of a headache; she could tell the physicians her name but little else, and became less responsive over time. Brain scans showed abnormal swelling and inflammation in several regions, with smaller areas where some cells had died.

Physicians diagnosed a dangerous condition called acute necrotizing encephalopathy, a rare complication of influenza and other viral infections.

“The pattern of involvement, and the way that it rapidly progressed over days, is consistent with viral inflammation of the brain,” Dr. Elissa Fory, a neurologist with Henry Ford Health System, said through an email. “This may indicate the virus can invade the brain directly in rare circumstances.” The patient is in critical condition.

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These domestic reports follow similar observations by doctors in Italy and other parts of the world, of Covid-19 patients having strokes, seizures, encephalitis-like symptoms and blood clots, as well as tingling or numbness in the extremities, called acroparesthesia. In some cases, patients were delirious even before developing fever or respiratory illness, according to Dr. Alessandro Padovani, whose hospital at University of Brescia in Italy opened a separate NeuroCovid unit to care for patients with neurological conditions.

The patients who come in with encephalopathy are confused and lethargic and may appear dazed, exhibiting strange behavior or staring off into space. They may be having seizures that require immediate medical care, and experts are warning health care providers who treat such patients to recognize that they may have Covid-19 and to take precautions to protect themselves from infection.

Eating three to six eggs a week could cut your risk of heart disease

What does your doctor say? I wouldn't necessarily trust the analysis, can the poorest people in China afford eggs?

Eating three to six eggs a week could cut your risk of heart disease

A new Chinese study has found that eating eggs several times a week could be linked with a lower risk of cardiovascular disease and death.
Carried out by researchers from Fuwai Hospital, Chinese Academy of Medical Sciences, the new study looked at 102,136 participants from 15 provinces across China, who were all free of cardiovascular disease (CVD) and cancer at the start of the study.
The participants completed food-frequency questionnaires to assess their egg consumption and were then followed for 17 years.
The findings, published in the journal Science China Life Sciences, showed that participants who ate three to six eggs per week had the lowest risk of CVD and death among the group.
However, a low or high intake of eggs appeared to be associated with a higher risk of CVD and death, with eating less than one egg a week linked with a 22 percent higher risk of CVD and a 29 percent higher risk of death, and eating ten or more eggs per week linked with a 30 percent higher risk of CVD and a 13 percent higher risk of death.
The researchers also found that egg consumption appeared to have a different effect on different types of CVD. While those who ate more eggs had a higher risk of coronary heart disease (CHD) and ischemic stroke, those who ate fewer eggs had a higher risk of hemorrhagic stroke.
Eggs are known to be a quick and affordable source of high-quality proteins, packed with nutrients that are beneficial for health. However, they are also high in cholesterol, meaning there has been some uncertainty as to whether consuming them could also increase cholesterol levels and the risk of CVD. The researchers say that up until now the findings from most studies looking at this association have been inconsistent, and no consensus has been reached about recommendations on egg intake. They also add that their findings highlight that moderate egg consumption of three to six eggs a week should be recommended for CVD prevention in China.
A large-scale US study published in The BMJ just last month also found that, despite previous concerns, eating an egg a day is not linked with a higher risk of CVD, compared to eating less than one egg per month. After investigating further and carrying out a meta-analysis of 28 observational studies, the team again failed to find a link between eating eggs and CVD risk among participants in the US and European studies.

Study Shows Choline Benefits for Your Brain RSS

Read and ask your doctor about this.

Study Shows Choline Benefits for Your Brain

A new study shows that taking in choline throughout your life helps keep your brain young and protects against Alzheimer's disease. Keep reading to find out more about the study, what choline is, its benefits, and how to get choline in your diet.
[Disclaimer: This post is not meant to be medical advice. I am summarizing and passing on information based on my research and experience. Check with your own medical providers for personalized guidance.]

What is Choline?

Choline is an essential nutrient. That means that you have to have it.
It was only added to the US list of required nutrients in 1998, so recognition of its importance is relatively new.
It is similar to B vitamins but it is not a vitamin.
Your body can produce a small amount of choline, but you need to eat or take in the bulk of it from diet or supplements.

Choline Benefits

Choline is vital to the structure of cells throughout your body and is of particular importance for liver and nervous system function. It helps lift mood, boost memory and energy, and reduce inflammation.
Adequate amounts of choline are important to your brain for several reasons.

Choline is used by your body to make fats and move fats to where they are needed. (Your brain cells are largely made up of fats, so this is a key nutrient for a healthy brain.)
It is used to make the neurotransmitter acetylcholine. Acetylcholine is used for muscle control, memory, and other brain functions.

Choline Research Study

Researchers at Arizona State University studied the effect of lifetime choline supplementation on mice who were bred to develop Alzheimer's-like symptoms (AD mice) and mice who did not have the Alzheimer's gene (non-AD mice) against control groups of both types of mice who were not supplemented.
The mice were repeatedly placed in a maze to test their memory. The AD mice that were given choline supplements throughout their life had better memory capability than AD control mice that were not supplemented.
Examining brain tissue from the mice showed less microglia activation in the supplemented groups for both AD and non-AD mice compared to their control groups. Activated microglia are associated with neurodegenerative diseases such as Alzheimer's, traumatic brain injury, multiple sclerosis, and Parkinson's disease.
The researchers concluded that their results suggest that "additional dietary choline may be an avenue to reduce brain inflammation in both neurodegenerative disease and the nondiseased aging brain."
To read the study: Velazquez R, Ferreira E, Knowles S, et al. Lifelong choline supplementation ameliorates Alzheimer’s disease pathology and associated cognitive deficits by attenuating microglia activation. Aging Cell. September 2019:e13037.

Recommended Amounts of Choline

The minimum amount of choline that you should be getting is the Recommended Adequate Intake (AI):

550 mg/day for adult males
425 mg/day for adult females

For maximizing the benefits of choline, you need to be getting more than the minimum.
There also is a Tolerable Upper Limit (UL) established for choline. It is recommended that you don't go over this amount with supplements:

3,500 mg/day

Choline and Folate

Choline and folate play well together. To optimize your use of choline, make sure you are getting enough folate also. (Folic acid is sometimes included in supplements instead of folate, but it is not really what your body needs. If you want to boost your folate using supplements, look for those that say folate on the nutrient breakdown.)

Choline Rich Food Sources

Choline can be found naturally in some foods. The best food sources of choline include:

pasture-raised eggs,
liver (chicken or beef),
grass-fed beef and pastured pork,
wild salmon,
dairy products, and
cruciferous vegetables (cauliflower, broccoli, cabbage, bok choy, kale, Brussel's sprouts).

These foods are also good sources of folate.

What is choline, Choline foods sources, Choline rich foods

Choline Supplements

Some experts recommend CDP choline, also called Citicoline, or Alpha GPC choline. When I looked on Amazon, they are either extremely expensive or have quite a lot of bad reviews where people are talking about experiencing negative side effects like headaches.
Personally, I have used Thorne Research's Phosphatidyl Choline for many years. (Thorne is a high-quality supplement company that stays away from GMO sources. This is particularly important with choline supplements as they are most often sourced in soybeans and most soybeans are now GMO.) It seems to me that my brain is sharper when I use it. I also eat many choline-rich foods.

Scientists Confirm: Herpes Virus Implicated in Alzheimer’s Disease

You'll want your doctor to know what to do with this to prevent your getting dementia.

Your chances of getting dementia.

1. A documented 33% dementia chance post-stroke from an Australian study? May 2012.

2. Then this study came out and seems to have a range from 17-66%. December 2013.

3. A 20% chance in this research. July 2013.

4. Dementia Risk Doubled in Patients Following Stroke September 2018

5. Parkinson’s Disease May Have Link to Stroke March 2017

The latest here:

Scientists Confirm: Herpes Virus Implicated in Alzheimer’s Disease

A coalition of scientists from the Icahn School of Medicine in New York City and Arizona State University, collected over 2,000 post-mortem tissue samples from 944 brains held at several brain banks funded by the US National Institute of Aging, which also provided money for the study. Some of the brain samples had the tell-tale amyloid plaques and tau buildups found in Alzheimer’s disease and some had signs of other cognitive impairments. Others were “control” patients with healthy brain function. What they discovered is groundbreaking news for scientists and any lives touched Alzheimer’s disease. They published their results in the science journal, Neuron, last week.
The herpes viruses
There are 9 herpes viruses known to man but when people think of herpes, they usually think of sexually transmitted diseases and unsightly cold sores erupting from infected lips. Those are the herpes viruses, HSV1 and HSV2. The peculiar thing about any herpes virus is its ability to go dormant in the body only to reawaken later and begin reproducing all over again causing diseases like shingles. In fact, the name herpes is derived from the Greek word meaning, “to creep” because of their ability to creep up nerves and plant themselves on our spines and in some instances, even our brains, probably having an effect on Alzheimer’s disease.
Although the research team found over 500 types of viruses present in those 2,000 brains, the two forms of herpes virus – HHVA6 and 7 – were found in particularly high concentrations in the brains of individuals with Alzheimer’s. After their dormant stage, they start actively reproducing and appear to speed up the Alzheimer’s-related protein build ups that ultimately lead to the brain’s system failure. Although not causative, they are described as “putting gas on the flame,” by Joel Dudley, a geneticist at the Icahn School of Medicine at Mt. Sinai in New York City, who described the virus as accelerating the disease. The study offers strong evidence that viral infections can influence its course.
Just an accident
Like the discovery of penicillin, many scientific discoveries are made by accident and this discovery is no different. The goal for this study was to identify new targets for drugs by using genetic data to look for differences between healthy brain tissue and brain tissue from those who had developed Alzheimer’s. Dudley says that, “Viruses were the last thing we were looking for.” But when they began to analyze the differences, “it just sort of came screaming out at us from the data.” The two human herpes viruses HHV6 and HHV7 were twice as high in brain tissue from people with Alzheimer’s!
Once they knew this, the next step was to find out how the viruses could affect the course of a brain disease. In order to do this, they set about identifying interactions between virus genes and other genes in the brains cells by mapping out a kind of social network between the two. Dudley wanted to know, “If the viruses are tweeting, who’s tweeting back?”
What they found were that the herpes virus genes were interacting with genes known to both increase a person’s risk for Alzheimer’s and to make a person’s brain more vulnerable to infection with HHV6 and HHV7. Just having the virus present in the brain isn’t enough to cause Alzheimer’s. Something needs to activate the virus out of its dormancy. To date, that activating factor has not been found but Dudley is hopeful and suspects some internal function of the brain cells is the cause. Still, because of the importance of this finding, we are two steps closer to finding a way to slow and maybe even stop the progression of the disease.

Research
1. Multiscale Analysis of Independent Alzheimer’s Cohorts Finds Disruption of Molecular, Genetic, and Clinical Networks by Human Herpesvirus. Neuron.
https://www.cell.com/neuron/fulltext/S0896-6273(18)30421-5
2. Herpes Viruses Implicated in Alzheimer’s Disease. The Scientist. June, 21, 2018
https://www.the-scientist.com/news-opinion/herpes-viruses-implicated-in-alzheimer-s-disease-64246
3. Researchers Find Herpes Viruses In Brains Marked By Alzheimer’s Disease. NPR. June 21, 2018.
https://www.npr.org/sections/health-shots/2018/06/21/621908340/researchers-find-herpes-viruses-in-brains-marked-by-alzheimers-disease