…transported in a regulated fashion. These processes are critical for synapse function and formation, programmed cell death mechanisms, and protection of mitochondrial DNA. Changes in mitochondrial dynamics are increasingly being linked to neurodegenerative diseases. However, these mitochondrial processes have been very difficult to study directly, particularly in the brain. Using novel methodology, Dr. Berman’s laboratory directly studies the role of mitochondrial dynamics in neurotoxicity/neuroprotection in chronic Parkinson’s disease models, aging, and other neurodegenerative diseases such as Alzheimer’s disease, with the goals of elucidating important mitochondrial mechanisms in neurodegeneration and providing potential new therapeutic targets.
Burton Lab
Edward A. Burton, MD, DPhil, FRCP
(1) α-Syn, mitochondria and Parkinson’s disease: We recently showed that short hairpin RNA targeting the SNCA gene encoding α-syn prevented motor deficits and neurodegeneration in a rat Parkinson’s disease model caused by exposure to a mitochondrial inhibitor, rotenone (Zharikov et al 2015). In our current studies, we are investigating the mechanisms underlying this observation and working to determine whether therapies targeting α-syn expression can be translated into effective neuroprotective treatments for Parkinson’s disease.
(2) Tauopathy: Several neurological disorders, including progressive supranuclear palsy (PSP), Alzheimer’s disease (AD), and chronic traumatic encephalopathy, are characterized pathologically by the accumulation of insoluble forms of Tau protein in neurons and glia. Collectively, tauopathies are common and incurable and there is an unmet need for treatments that target pathogenic mechanisms. We are currently developing zebrafish models of tauopathy that replicate key aspects of human disease, and which are optimized for use in drug discovery and development (Bai et al 2007, 2011a, and unpublished).
(3) Tools for high-throughput neurobehavioral phenotyping in zebrafish models: We have developed technology to carry out high-throughput measurement of neurobehavioral phenotypes in zebrafish disease models in 96-well plates (Cario et al 2011; Zhou et al 2014). We are currently investigating how this approach can be used for chemical modifier screens for drug discovery applications in disease models. We are also developing new tools to allow automated quantification of other neurological phenotypes.
(4) Dystonia: Primary torsion dystonia, caused by mutation of the DYT1 gene encoding torsinA, is characterized clinically by generalized, involuntary and sustained muscle contractions resulting in abnormal twisting movements and postures. Although the etiology of DYT1 dystonia is well-defined genetically, the pathophysiology is relatively poorly understood. We have characterized the DYT1 orthologue in zebrafish (Sager et al 2012) and are currently constructing zebrafish dystonia models to allow elucidation of the abnormal neuronal biology and circuitry formation underlying dystonia.
(5) Oligodendroglia and axonal regeneration: There has been increasing recognition that oligodendrocytes have important roles in maintenance of axonal viability, in addition to production of the myelin sheath. Using the zebrafish as a vertebrate model in which axons regenerate robustly, we have embarked on studies to identify the molecular pathways by which glia can support axonal growth in the CNS. This work involves novel zebrafish models that express axonal injury-responsive transgenes in oligodendrocytes (Bai et al 2011b, 2014).
Chu Laboratory
Charleen T. Chu, MD, PhD
My laboratory studies neuronal autophagy, protein kinases and mitochondrial pathobiology, with an emphasis on genetic and toxin models of Parkinson’s disease. I have >26 years of research experience with cell signaling, proteolysis and post-translational protein modifications, with additional expertise in immunochemical image analysis as a practicing neuropathologist. A central area of interest involves the regulation of mitochondrial function and turnover, studied with respect to: 1) toxins that affect mitochondrial function, and 2) kinases whose mutations cause familial Parkinson’s disease. Using cell biologic, molecular imaging and mass spectrometry approaches, my team identified novel phosphorylation sites of the major autophagy protein LC3, and of the mitochondrial transcription factor A (TFAM A), which modulate dendritic/post-synaptic neuron injury. We also discovered a basic mechanism by which damaged mitochondria are recognized by the autophagy system for clearance, based on redistribution of the inner membrane phospholipid cardiolipin. Current efforts are directed at the interplay of aging and calcium dysregulation in the LRRK2 model, mechanisms that regulate PINK1 processing and the subcellular compartmentalization of its function in neurons, and transcriptional (mtDNA and nDNA) mechanisms that underlie the catabolic-anabolic imbalance leading to neurodegeneration.
Hastings Laboratory
Teresa G. Hastings, PhD
Dr. Hastings’ research focuses on the pathogenesis and treatment of neurodegenerative diseases. Specifically, her work examines the role of oxidative stress in the selective vulnerability of dopaminergic neurons in Parkinson’s disease. Under oxidative conditions, the catechol ring of dopamine will oxidize spontaneously or via enzymatic mechanisms to form reactive oxygen species and dopamine quinones. These metabolites of dopamine will covalently modify cellular proteins and thus have the potential to induce cytotoxicity. Dr. Hastings’ laboratory has shown that injections of dopamine into the striatum of rats leads to the formation of protein cysteinyl-catechols, a product of dopamine oxidation, and results in selective toxicity to dopamine terminals. Questions currently being examined in her laboratory include what mechanisms are involved in dopamine-induced selective toxicity, the effects of dopamine on mitochondrial dysfunction and using proteomic techniques, what are the proteins modified by dopamine quinones to induce dopaminergic cell death. Other research interests of Dr. Hastings include the role of dopamine, oxidative stress, and mitochondrial dysfunction in the mechanism of methamphetamine-induced toxicity to dopamine terminals. A variety of molecular and immunohistochemical approaches in both in vitro and in vivo models are used in this work.
Postmortem
- Events process
- Moments after death, the brain is placed in a condition of asphyxia, and tissues are deprived of oxygen, which is required as the final electron acceptor at the terminus of the electron transport chain. → mitochondrial ATP generation ceases. → destruction of cellular membranes (19), because phospholipids in the membranes become rapidly oxidized and are not replaced by the ATP-dependent processes of membrane repair and renewal.
- Heterogeneity
- Factors
- age, drug use, and possible brain infections, as well as cause of death, storage condition of the body, tissue pH, and PMI
- Factors
Sample preparation
| Frozen | FFPE | |
|---|---|---|
| DNA, RNA PTM | Preferable | (the variability that nonstandardized preparation methods introduce into FFPE samples affects the integrity of molecular data disproportionately. "For example, some users leave a tissue biopsy in formalin for a longer period of time, which might not affect immunostaining much but is detrimental for any further molecular analysis," says Michael Kazinski, senior director of molecular preanalytic technologies in sample technologies marketing at QIAGEN. Long DNA fragments, protein phosphorylation states and other PTMs are rarely, if ever, preserved in FFPE samples, for instance. And, because formalin inactivates neither RNases nor phosphatases, FFPE samples often contain degraded RNA and non-native configurations of phosphorylated proteins.) It is known that DNA in FFPE tissue accumulates sequence artifacts that can lead to false results in sequencing experiments," says Kazinski. "Those artifacts do not occur in frozen tissue." |
| mass spectrometry, quantitative real-time PCR, next-generation sequencing and Western blotting, ELISA (도 겠지, lysate 쓰니까) | Necessity, Frozen sections are considered the gold standard for most molecular assays, especially [for sequencing DNA or RNA] strands that are longer than 50 base pairs," says Jennifer Freeland, research and development manager at Thermo Fisher Scientific (which recently acquired Life Technologies). Also, Freeland says, "frozen tissue is best when experimenting with new antibodies and immunohistochemistry results are unknown." | |
| immunostaining | Doable (cryosection → OCT/Superfrost → dry → store at -80) | Best |
| Radioligand binding assay | O |
Factors
| Factors | Index | ||
|---|---|---|---|
| Pre-mortem | Metabolic state | pH (neutral is good, acidosis is bad) | |
| hypoxia | pH (neutral is good, acidosis is bad) | ||
| Post-mortem | Toxic substance and drug | ||
| infection | |||
| Seizure | |||
| Interval | |||
| Temperature (before cryopreservation at -80) | Js: This is more important. (Ferrer, 2007 #2049) 10c: stable up to 50h, 4oC and room temperature: modfied | ||
Proteins on Postmortem delay (PMI)
- Overall conclusion
- Preservation of the samples at room temperature for more than 12 hours makes the biochemical study less reliable.
- As a marginal note, thawed tissue stored at room temperature and then refrozen is definitely no longer usable for biochemical studies.
아래 표는 대개 (Ferrer, 2007 #2049)
| Resistant proteins (up to 50 h)/mRNA | Vulnerable proteins/mRNA (temperature for 24 h) |
|---|---|
| Synaptophysin, PSD-95 | (normal) Syn |
| Cathepsin D | ATP synthase (room temperature for 24 h) |
| Proteasome subunits (and proteasomal activity) | Phosphor-tau |
| IL-6 positive count of glial cells (Trautz 2019) | Syntaxin |
| mRNA expression levels of IL-1β gene of the brain and adrenal gland tissues showed significant time-dependent reductions began at 6 h till 48 h postmortem. (Dalia Mohamed Ali 2018) |
- Utility of PM tissues
- Needs alignment within NSTM on the utility/limitation of PM tissues for translational research
- Major challenging question: Protein preservation is affected by autopsy conditions - PMI (postmortem interval) and pH (metabolic disturbances). So, isn’t PM study useless? Or, can we set a general criteria for inclusion in research?
- → The degree of protein preservation largely varies among individual proteins in Human.
- Some proteins (e.g. synaptophysin, proteasome components, cathepsin D) are stable postmortem up to 50 h, whereas others are not (e.g. syn, ATP-synthase) (Ferrer et al. 2007, PMID 17204935).
- It may be difficult to set a criteria for a specific protein unless we actually characterize the protein of interest.
- Proposal: Small PMI and neutral pH are preferrable. But a PM study can still be successfully designed as long as these variables are controlled across disease group and control group.
Postural instability
| prototype | Currently, there are no validated digital tools for the assessment of Falls in PD; however, there are validated digital tools for measuring biomarkers that are correlated to risk of falls. Fall risk is correlated with several aspects of gait. Two of these aspects, stride and step time variability, are variables that can be measured with high precision using digital gait assessment tools in the clinic, and these digital gait assessment tools have been clinically validated against gold-standard systems specifically in PD patients. As such, use of gait variability is a more appropriate endpoint for studies than actual falls, given that falls are a relatively rare occurrence (even among PD patients). In the TAK071-2002 study, an in-clinic digital gait assessment tool is being used to measure the primary endpoint of gait variability. The results of this study will indicate if the digital tool is fit-for- |
Uncertain Spans
- The first sentence at the very top of the page wraps from the previous photo and starts mid-sentence; the words preceding “transported in a regulated fashion” are not visible on this photo.
- Several inner cells of the Pre-mortem / Post-mortem Factors table are visually empty in the source; whether they are intentionally blank or partially obscured by table grid lines could not be determined unambiguously.