Teasing Out Purkinje Proteins

Scientists have developed a method of extracting Purkinje cells from the brain, identifying potential Purkinje-specific protein markers in the process.

AsianScientist (Jun 9, 2014) – A team of scientists from Japan has identified a catalog of proteins specific to Purkinje neurons, a roadmap that could reveal insights into diseases such as autism and ataxias. The study has been published in the journal Genome Research.

Purkinje neurons are a type of brain cell found in the cerebellum, which plays a key role in activating and coordinating muscular movement. They contain many dendrites, which are branched extensions that receive signals, and a single axon, which transmits signals to other neurons. Due to their extensive distribution and close intermingling with other types of brain cells, examining the contents of dendrites is known to be difficult.

A team led by Dr. Thomas Launey of RIKEN’s Brain Sciences Institute and Dr. Charles Plessy of RIKEN’s Life Science Technologies has now successfully identified the proteins that distinguish Purkinje cells from other neurons.

Firstly, they used a modified adeno-associated virus (AAV) to specifically target Purkinje cells, introducing a fluorescent tag which labelled ribosomal proteins. The strong fluorescence of the introduced construct allowed the researchers to perform microdissection, separating the Purkinje cell body from the dendrites.

The team then analysed the RNA sequences obtained by combining two “high-throughput” methods known as TRAP (Translating Ribosomal Affinity Purification) and CAGEscan (Cap Analysis of Gene Expression). In TRAP, the RNA sequences associated with the ribosomes are separated out from each sample by isolating the labelled ribosomal proteins. CAGEscan, a technology developed by RIKEN, then allowed them to quickly sequence and identify the huge number of trapped mRNAs.

“The introduction of new methods gave us access to mRNAs that are often difficult to detect, especially those that are in low abundance, but are functionally important, such as membrane receptors, transporters and ion channels,” said Dr. Launey.

The near-exhaustive purification method also allowed the researchers to identify Purkinje cell-specific genes and transcription factor binding sites that could help explain the cell’s function.

The methods used in this study could be widely applicable to other neuron types and other mammals, including primates, paving the way for comparative studies that may reveal the origins of complex cognitive abilities or the causal defects in diseases affecting the brain.

The article can be found at: Kratz et al. (2014) Digital expression profiling of the compartmentalized translatome of Purkinje neurons.

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Source: RIKEN; Photo: Giulio Callegaro/CC/Flickr.
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Alan Aw is a maths enthusiast who likes sharing the fun and beauty of science with others. Besides reading, he enjoys running, badminton, and listening to (and occasionally playing) Bach or Zez Confrey.

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