DMPK mRNA Expression in Human Brain Tissue Throughout the Lifespan

ObjectiveMyotonic dystrophy is a multisystem disorder caused by a trinucleotide repeat expansion on the myotonic dystrophy protein kinase (DMPK) gene. To determine whether wildtype DMPK expression patterns vary as a function of age, we analyzed DMPK expression in the brain from 99 donors ranging from 5 postconceptional weeks to 80 years old.MethodsWe used the BrainSpan messenger RNA sequencing and the Yale Microarray data sets, which included brain tissue samples from 42 and 57 donors, respectively. Collectively, donors ranged in age from 5 postconceptional weeks to 80 years old. DMPK expression was normalized for each donor across regions available in both data sets. Restricted cubic spline linear regression models were used to analyze the effects of log-transformed age and sex on normalized DMPK expression data.ResultsAge was a statistically significant predictor of normalized DMPK expression pattern in the human brain in the BrainSpan (p < 0.005) and Yale data sets (p < 0.005). Sex was not a significant predictor. Across both data sets, normalized wildtype DMPK expression steadily increases during fetal development, peaks around birth, and then declines to reach a nadir around age 10.ConclusionsPeak expression of DMPK coincides with a time of dynamic brain development. Abnormal brain DMPK expression due to myotonic dystrophy may have implications for early brain development.

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Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies

Biochemical Journal ◽

10.1042/bj20121742 ◽

2013 ◽

Vol 453 (1) ◽

pp. 101-113 ◽

Cited By ~ 54

Author(s):

Paul Davies ◽

Kelly M. Hinkle ◽

Nour N. Sukar ◽

Bryan Sepulveda ◽

Roxana Mesias ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Human Brain ◽

Brain Tissue ◽

Kinase Activity ◽

Expression Patterns ◽

Significant Proportion ◽

Leucine Rich Repeat ◽

Human Brain Tissue ◽

Robust Detection ◽

Lrrk2 Protein

Missense mutations in LRRK2 (leucine-rich repeat kinase 2) are a major cause of PD (Parkinson's disease). Several antibodies against LRRK2 have been developed, but results using these polyclonal antibodies have varied widely leading to conflicting conclusions. To address this challenge, the Michael J. Fox Foundation for Parkinson's Research generated a number of monoclonal antibodies targeting epitopes across the LRRK2 protein. In the present paper, we report optimized protocols and results for ten monoclonal antibodies for immunoblotting, immunohistochemistry, immunoprecipitation and kinase activity assays, in rat, mouse and human brain tissue. Several efficacious antibodies were identified, but results demonstrate that the mouse monoclonal N241A/34 is suitable for most applications, with the best overall rabbit monoclonal antibody being c41-2. These antibodies produced a dominant band of the expected size via immunoblotting and a lack of labelling in tissue derived from LRRK2-knockout animals under optimized conditions. A significant proportion of LRRK2 protein localizes to insoluble fractions and no evidence of truncated LRRK2 protein was detected in any fraction from rodent or human tissues. An assay was developed for the robust detection of LRRK2 kinase activity directly from frozen mouse and human brain tissue, but precipitous declines in activity were observed that corresponded to increasing post-mortem intervals and processing times. Finally, we demonstrate the highest levels of brain-localized LRRK2 in the striatum, but note differential expression patterns between rat and mouse in both striatum and cortex. Anti-LRRK2 monoclonal antibodies that are unlimited in availability together with the proposed standardized protocols should aid in the definition of LRRK2 function in both health and disease.

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Recovery and Expression of Messenger RNA from Postmortem Human Brain Tissue

Modern Pathology ◽

10.1038/modpathol.3880451 ◽

2001 ◽

Vol 14 (11) ◽

pp. 1157-1161 ◽

Cited By ~ 54

Author(s):

Thomas J Cummings ◽

Jay C Strum ◽

Lawrence W Yoon ◽

Mari H Szymanski ◽

Christine M Hulette

Keyword(s):

Human Brain ◽

Brain Tissue ◽

Messenger Rna ◽

Human Brain Tissue ◽

Postmortem Human Brain

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RBIO-06. IMPLEMENTATION OF HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED CEREBRAL ORGANOIDS TO MODEL NORMAL TISSUE RADIORESPONSE

Neuro-Oncology ◽

10.1093/neuonc/noaa215.806 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii193-ii193

Author(s):

Lawrence Bronk ◽

Sanjay Singh ◽

Riya Thomas ◽

Luke Parkitny ◽

Mirjana Maletic-Savatic ◽

...

Keyword(s):

Stem Cell ◽

Human Brain ◽

Brain Tissue ◽

Human Tissue ◽

Model Systems ◽

Human Brain Tissue ◽

Post Irradiation ◽

Mature Neurons ◽

Induced Pluripotent ◽

Effects Of Radiation

Abstract Treatment-related sequelae following cranial irradiation have life changing impacts for patients and their caregivers. Characterization of the basic response of human brain tissue to irradiation has been difficult due to a lack of preclinical models. The direct study of human brain tissue in vitro is becoming possible due to advances in stem cell biology, neuroscience, and tissue engineering with the development of organoids as novel model systems which enable experimentation with human tissue models. We sought to establish a cerebral organoid (CO) model to study the radioresponse of normal human brain tissue. COs were grown using human induced pluripotent stem cells and a modified Lancaster protocol. Compositional analysis during development of the COs showed expected populations of neurons and glia. We confirmed a population of microglia-like cells within the model positive for the makers Iba1 and CD68. After 2-months of maturation, COs were irradiated to 0, 10, and 20 Gy using a Shepard Mark-II Cs-137 irradiator and returned to culture. Subsets of COs were prepared for immunostaining at 30- and 70-days post-irradiation. To examine the effect of irradiation on the neural stem cell (NSC) population, sections were stained for SOX2 and Ki-67 expression denoting NSCs and proliferation respectively. Slides were imaged and scored using the CellProfiler software package. The percentage of proliferating NSCs 30-days post-irradiation was found to be significantly reduced for irradiated COs (5.7% (P=0.007) and 3.4% (P=0.001) for 10 and 20 Gy respectively) compared to control (12.7%). The reduction in the proliferating NSC population subsequently translated to a reduced population of NeuN-labeled mature neurons 70 days post-irradiation. The loss of proliferating NSCs and subsequent reduction in mature neurons demonstrates the long-term effects of radiation. Our initial results indicate COs will be a valuable model to study the effects of radiation therapy on normal and diseased human tissue.

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