David B. Berry

Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism

Significance Prions are proteins that assume alternate shapes that become self-propagating, and while some prions perform normal physiological functions, others cause disease. Prions were discovered while studying the cause of rare neurodegenerative diseases of animals and humans called scrapie and Creutzfeldt–Jakob disease, respectively. We report here the discovery of α-synuclein prions that cause a more common neurodegenerative disease in humans called multiple system atrophy (MSA). In contrast to MSA, brain extracts from Parkinson’s disease (PD) patients were not transmissible to genetically engineered cells or mice, although much evidence argues that PD is also caused by α-synuclein, suggesting that this strain (or variant) is different from those that cause MSA. Prions are proteins that adopt alternative conformations that become self-propagating; the PrPSc prion causes the rare human disorder Creutzfeldt–Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T–YFP) and TgM83+/− mice expressing α-synuclein (A53T). The TgM83+/− mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83+/+ mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83+/− mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T–YFP in cultured cells, whereas none of six Parkinson’s disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83+/+ mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.

Multiple Means to the Same End: The Genetic Basis of Acquired Stress Resistance in Yeast

In nature, stressful environments often occur in combination or close succession, and thus the ability to prepare for impending stress likely provides a significant fitness advantage. Organisms exposed to a mild dose of stress can become tolerant to what would otherwise be a lethal dose of subsequent stress; however, the mechanism of this acquired stress tolerance is poorly understood. To explore this, we exposed the yeast gene-deletion libraries, which interrogate all essential and non-essential genes, to successive stress treatments and identified genes necessary for acquiring subsequent stress resistance. Cells were exposed to one of three different mild stress pretreatments (salt, DTT, or heat shock) and then challenged with a severe dose of hydrogen peroxide (H2O2). Surprisingly, there was little overlap in the genes required for acquisition of H2O2 tolerance after different mild-stress pretreatments, revealing distinct mechanisms of surviving H2O2 in each case. Integrative network analysis of these results with respect to protein–protein interactions, synthetic–genetic interactions, and functional annotations identified many processes not previously linked to H2O2 tolerance. We tested and present several models that explain the lack of overlap in genes required for H2O2 tolerance after each of the three pretreatments. Together, this work shows that acquired tolerance to the same severe stress occurs by different mechanisms depending on prior cellular experiences, underscoring the context-dependent nature of stress tolerance.

Drug resistance confounding prion therapeutics

Significance As people live longer, the prevalence and economic impact of neurodegenerative diseases rise. No cures or effective treatments exist for any of these fatal disorders, so identifying potential therapeutics that extend survival in animal models is vital. Many neurodegenerative illnesses have been shown to be caused by the accumulation of self-propagating misfolded proteins—the hallmark of prion diseases. We report the efficacy of 2-aminothiazoles, which were identified in cell-based screens as antiprion compounds, in extending the lives of prion-infected animals. Efficacy was limited by the development of drug-resistant prions, which is likely to have important implications for creating therapeutics in many different neurodegenerative diseases. There is not a single pharmaceutical that halts or even slows any neurodegenerative disease. Mounting evidence shows that prions cause many neurodegenerative diseases, and arguably, scrapie and Creutzfeldt–Jakob disease prions represent the best therapeutic targets. We report here that the previously identified 2-aminothiazoles IND24 and IND81 doubled the survival times of scrapie-infected, wild-type mice. However, mice infected with Rocky Mountain Laboratory (RML) prions, a scrapie-derived strain, and treated with IND24 eventually exhibited neurological dysfunction and died. We serially passaged their brain homogenates in mice and cultured cells. We found that the prion strain isolated from IND24-treated mice, designated RML[IND24], emerged during a single passage in treated mice. Although RML prions infect both the N2a and CAD5 cell lines, RML[IND24] prions could only infect CAD5 cells. When passaged in CAD5 cells, the prions remained resistant to high concentrations of IND24. However, one passage of RML[IND24] prions in untreated mice restored susceptibility to IND24 in CAD5 cells. Although IND24 treatment extended the lives of mice propagating different prion strains, including RML, another scrapie-derived prion strain ME7, and chronic wasting disease, it was ineffective in slowing propagation of Creutzfeldt–Jakob disease prions in transgenic mice. Our studies demonstrate that prion strains can acquire resistance upon exposure to IND24 that is lost upon passage in mice in the absence of IND24. These data suggest that monotherapy can select for resistance, thus intermittent therapy with mixtures of antiprion compounds may be required to slow or stop neurodegeneration.

Pathway connectivity and signaling coordination in the yeast stress‐activated signaling network

Stressed cells coordinate a multi‐faceted response spanning many levels of physiology. Yet knowledge of the complete stress‐activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho‐proteome changes in cells responding to salt stress, to infer the salt‐responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown ‘hubs’ of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress‐defense genes with reduction of growth‐related transcripts. We find that the orthologous human network is enriched for cancer‐causing genes, underscoring the importance of the subnetworks predictions in understanding stress biology.

Contribution of Lumbar Spine Pathology and Age to Paraspinal Muscle Size and Fatty Infiltration

Study Design. Retrospective chart analysis of 199 individuals aged 18 to 80 years scheduled for lumbar spine surgery. Objective. The purpose of this study was to quantify changes in muscle cross-sectional area (CSA) and fat signal fraction (FSF) with age in men and women with lumbar spine pathology and compare them to published normative data. Summary of Background Data. Pathological changes in lumbar paraspinal muscle are often confounded by age-related decline in muscle size (CSA) and quality (fatty infiltration). Individuals with pathology have been shown to have decreased CSA and fatty infiltration of both the multifidus and erector spinae muscles, but the magnitude of these changes in the context of normal aging is unknown. Methods. Individuals aged 18 to 80 years who were scheduled for lumbar surgery for diagnoses associated with lumbar spine pain or pathology were included. Muscle CSA and FSF of the multifidus and erector spinae were measured from preoperative T2-weighted magnetic resonance images at the L4 level. Univariate and multiple linear regression analyses were performed for each outcome using age and sex as predictor variables. Statistical comparisons of univariate regression parameters (slope and intercept) to published normative data were also performed. Results. There was no change in CSA with age in either sex (P > 0.05), but women had lower CSAs than men in both muscles (P < 0.0001). There was an increase in FSF with age in erector spinae and multifidus muscles in both sexes (P < 0.0001). Multifidus FSF values were higher in women with lumbar spine pathology than published values for healthy controls (P = 0.03), and slopes tended to be steeper with pathology for both muscles in women (P < 0.08) but not in men (P > 0.31). Conclusion. Lumbar muscle fat content, but not CSA, changes with age in individuals with pathology. In women, this increase is more profound than age-related increases in healthy individuals. Level of Evidence: 3

Different 2-Aminothiazole Therapeutics Produce Distinct Patterns of Scrapie Prion Neuropathology in Mouse Brains

Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from ∼120 days to over 450 days. In addition to IND24, we evaluated the pharmacokinetics and efficacy of five additional 2-AMTs; one was not followed further because its brain penetration was poor. Of the remaining four new 2-AMTs, IND114338 doubled and IND125 tripled the incubation times of RML-inoculated wt and Tg4053 mice overexpressing wt mouse prion protein (PrP), respectively. Neuropathological examination of the brains from untreated controls showed a widespread deposition of self-propagating, β-sheet-rich “scrapie” isoform (PrPSc) prions accompanied by a profound astrocytic gliosis. In contrast, mice treated with 2-AMTs had lower levels of PrPSc and associated astrocytic gliosis, with each compound resulting in a distinct pattern of deposition. Notably, IND125 prevented both PrPSc accumulation and astrocytic gliosis in the cerebrum. Progressive central nervous system dysfunction in the IND125-treated mice was presumably due to the PrPSc that accumulated in their brainstems. Disappointingly, none of the four new 2-AMTs prolonged the lives of mice expressing a chimeric human/mouse PrP transgene inoculated with Creutzfeldt-Jakob disease prions.

Optimization of Aryl Amides that Extend Survival in Prion-Infected Mice

Developing therapeutics for neurodegenerative diseases (NDs) prevalent in the aging population remains a daunting challenge. With the growing understanding that many NDs progress by conformational self-templating of specific proteins, the prototypical prion diseases offer a platform for ND drug discovery. We evaluated high-throughput screening hits with the aryl amide scaffold and explored the structure–activity relationships around three series differing in their N-aryl core: benzoxazole, benzothiazole, and cyano. Potent anti-prion compounds were advanced to pharmacokinetic studies, and the resulting brain-penetrant leads from each series, together with a related N-aryl piperazine lead, were escalated to long-term dosing and efficacy studies. Compounds from each of the four series doubled the survival of mice infected with a mouse-passaged prion strain. Treatment with aryl amides altered prion strain properties, as evidenced by the distinct patterns of neuropathological deposition of prion protein and associated astrocytic gliosis in the brain; however, none of the aryl amide compounds resulted in drug-resistant prion strains, in contrast to previous studies on compounds with the 2-aminothiazole (2-AMT) scaffold. As seen with 2-AMTs and other effective anti-prion compounds reported to date, the novel aryl amides reported here were ineffective in prolonging the survival of transgenic mice infected with human prions. Most encouraging is our discovery that aryl amides show that the development of drug resistance is not an inevitable consequence of efficacious anti-prion therapeutics.

Bioassays and Inactivation of Prions

The experimental study of prions requires a model for their propagation. However, because prions lack nucleic acids, the simple techniques used to replicate bacteria and viruses are not applicable. For much of the history of prion research, time-consuming bioassays in animals were the only option for measuring infectivity. Although cell models and other in vitro tools for the propagation of prions have been developed, they all suffer limitations, and animal bioassays remain the gold standard for measuring infectivity. A wealth of recent data argues that both β-amyloid (Aβ) and tau proteins form prions that cause Alzheimers disease, and α-synuclein forms prions that cause multiple system atrophy and Parkinsons disease. Cell and animal models that recapitulate some of the key features of cell-to-cell spreading and distinct strains of prions can now be measured.

An Endplate-Based Joint Coordinate System for Measuring Kinematics in Normal and Abnormally-Shaped Lumbar Vertebrae.

Vertebral level-dependent, angular, and linear translations of the spine have been measured in 2D and 3D using several imaging methods to quantify postural changes due to loading conditions and tasks. Here, we propose and validate a semiautomated method for measuring lumbar intervertebral angles and translations from upright MRI images using an endplate-based, joint coordinate system (JCS). This method was validated using 3D printed structures, representing intervertebral discs (IVD) at predetermined angles and heights, which were positioned between adjacent cadaveric vertebrae as a gold standard. Excellent agreement between our measurements and the gold standard was found for intervertebral angles in all anatomical planes (ICC > .997) and intervertebral distance measurements (ICC > .949). The proposed endplate-based JCS was compared with the vertebral body-based JCS proposed by the International Society of Biomechanics (ISB) using the 3D printed structures placed between 3 adjacent vertebrae from a cadaver with scoliosis. The endplate-based method was found to have better agreement with angles in the sagittal plane (ICC = 0.985) compared with the vertebral body-based method (ICC = .280). Thus, this method is accurate for measuring 3D intervertebral angles in the healthy and diseased lumbar spine.

Lumbar spine postures in Marines during simulated operational positions

Low back pain has a 70% higher prevalence in members of the armed forces than in the general population, possibly due to the loads and positions soldiers experience during training and combat. Although the influence of heavy load carriage on standing lumbar spine posture in this population is known, postures in other operationally relevant positions are unknown. Therefore, the purpose of this study was to characterize the effect of simulated military operational positions under relevant loading conditions on global and local lumbar spine postures in active duty male US Marines. Secondary objectives were to evaluate if intervertebral disc degeneration and low back pain affect lumbar spine postures. Magnetic resonance images were acquired on an upright scanner in the following operational positions: Natural standing with no external load, standing with body armor (11.3 kg), sitting with body armor, and prone on elbows with body armor. Custom software was used to measure global lumbar spine posture: Lumbosacral flexion, sacral slope, lordosis, local measures of intervertebral angles, and intervertebral distances. Sitting resulted in decreased lumbar lordosis at all levels of the spine except L1–L2. When subjects were prone on elbows, a significant increase in local lordosis was observed only at L5–S1 compared with all other positions. Marines with disc degeneration (77%) or history of low back pain (72%) had decreased lumbar range of motion and less lumbar extension than healthy Marines. These results indicate that a male Marines pathology undergoes a stereotypic set of postural changes during functional tasks, which may impair performance.