Yale Scientists Say AAV5 is Best for Transduction in Primates

Scientists who research genetic disorders might want to reconsider the way they transfer therapeutic genes to cells in the central nervous system.

Adeno-associated virus (AAV) vectors are the most common way to transfer DNA into neural cells because of their nonpathogenic nature and long-term transduction abilities. Specifically, researchers most frequently use AAV serotype 2 (AAV2). But scientists at the Yale University School of Medicine believe that AAV serotype 5 (AAV5) is a more effective way to go when carrying out genetic transduction in the nonhuman primate brain.

In their study, the research team injected the brains of St. Kitts green monkeys with vectors from AAV serotypes 1-6, each of which contained an enhanced green-fluorescent protein reporter gene (GFP). One month later, immunohistochemistry and unbiased stereology were used to analyze the tissue and count the number of GFP cells.

“We used Stereo Investigator to do all of the quantitative comparisons between the different vector serotypes and their gene expression,” explained Dr. Gene Redmond.

Though all six serotypes were able to generate the reporter gene in the monkey’s brain cells, AAV serotype 5 was by far the most efficient, according to the study.

“The goal in human neurosurgical procedures delivering viral vectors to the brain is to make as little perturbation of brain tissue as is possible while delivering the needed gene for the appropriate duration. Viral vectors that can generate a large overall area of transduction and have a tropism for the desired cell type may allow for the delivery of the smallest possible amount of vector, resulting in maximal gene delivery while minimizing tissue damage, inappropriate spread, or the possibility of incorporation into the host genome.” (from the paper)

Read the free abstract or download the complete paper “Comparative Transduction Efficiency of AAV Vector Serotypes 1–6 in the Substantia Nigra and Striatum of the Primate Brain” at nature.com.

Eleni A Markakis, Kenneth P Vives, Jeremy Bober, Stefan Leichtle, Csaba Leranth, Jeff Beecham, John D Elsworth, Robert H Roth, R Jude Samulski and D Eugene Redmond Jr, “Comparative Transduction Efficiency of AAV Vector Serotypes 1–6 in the Substantia Nigra and Striatum of the Primate Brain” (Molecular Therapy (2010) 18 3, 588–593)

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Mount Sinai Scientists Research the Effect of Aging on Learning and Memory with Stereo Investigator

How do you find the volume of a sphere? It is a question that you’ve answered on math exams stretching all the way back to the sixth grade, but the formula eludes your brain. What is it that causes you to forget something that has been ingrained in your brain

Dr. John Morrison of Mount Sinai School of Medicine and Peter R. Rapp, PHD and other researchers have determined the specific types of nerve cells that diminish with age. With the help of Stereo Investigator, they’ve seen that these nerve cells—spines–play an important role in the learning process within the brain. As a person ages, the number of spines decreases in the brain, and that makes it all the more difficult to both learn and retain new information.

By administering delayed response testing to rhesus monkeys the team started to understand their learning skills. These tests challenged the monkeys’ memory by delayed non-matching-to-sample and delayed response tasks. Increasing the amount of time a reward was hidden challenged the monkeys’ memories. When the reward was hidden for longer periods of time, older monkeys showed slower response times compared to the younger monkeys.

Once the delayed response testing portion of the study was complete, Dr. Morrison explained that he and his team used Stereo Investigator to calculate the volume of cortical area 46 in the monkey’s brains. “We use [MBF Bioscience] software often to trace loaded neurons,” he added.

Dani Dumitriu, Jiandong Hao, Yuko Hara, Jeffrey Kaufmann, William G. M. Janssen, Wendy Lou, Peter R. Rapp, John H. Morrison, “Selective Changes in Thin Spine Density and Morphology in Monkey Prefrontal Cortex Correlate with Aging-Related Cognitive Impairment” (The Journal of Neuroscience, June 2, 2010, 30(22):7507–7515

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Stereo Investigator Helps Washington D.C. Scientists Study the Emergence of Human Language

History is being made at George Washington University’s Laboratory for Evolutionary Neuroanatomy, and Stereo Investigator is playing a part. Using Stereo Investigator to count neurons, estimate axon fiber length, and quantify cellular volumes, Dr. Chet C. Sherwood and his team are carrying out “detailed comparisons of neural phenotypes between humans and our closest relatives, the great apes.”

A recent focus at the lab is the emergence and evolution of the human language. In their paper “Wernicke’s area homologue in chimpanzees (Pan troglodytes) and its relation to the appearance of modern human language” (Proceedings of the Royal Society B: Biological Sciences), the authors concluded that the function of Wernicke’s area, a part of the brain linked to speech, and its location in the brain’s left hemisphere “evolved long before the emergence of modern human language.”

Using design based stereological methods, the researchers examined area Tpt in the Wernicke’s area of 12 chimpanzee brains. They used Stereo Investigator to manually draw area Tpt’s boundaries with a Zeiss Axioplan 2 microscope, and found that the number of neurons was greatest on the left side. These findings led them to determine that the positioning of Wernicke’s area originated before humans, chimpanzees, and bonobos evolved from their last common ancestor.

“Stereo Investigator is one of our most important tools,” said Dr. Sherwood. “The software is both intuitive and powerful.”

Read the abstract (free) and full article (by subscription) at royalsocietypublishing.org.

Muhammad A. Spocter, William D. Hopkins, Amy R. Garrison, Amy L. Bauernfeind, Cheryl D. Stimpson, Patrick R. Hof, and Chet C. Sherwood, “Wernicke’s area homologue in chimpanzees (Pan troglodytes) and its relation to the appearance of modern human language” Proceedings of the Royal Society B: Biological Sciences, 2010, doi: 10.1098/rspb.2010.0011

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Swiss Scientists Use Stereo Investigator to Study Schizophrenia

There may be more evidence that schizophrenia results from a combination of genetic and environmental factors. One of these hereditary influences may be an impaired ability to synthesize the antioxidant glutathione (GSH), which results in oxidative stress, according to a study conducted by scientists at the University of Lausanne in Switzerland.

By observing mice with a GSH deficit, Dr. Kim Q. Do and her team determined that the inability to synthesize GSH led to “inadequate responses to stress and fear,” while the capacity for spatial learning and spatial memory remained intact. Thus, they concluded that a deficiency of GSH results in a “selective decrease of PV-IR interneurons in CA3 and dentate gyrus (DG) of the ventral but not dorsal hippocampus.”

The research group quantified the density of GABAergic interneurons in various subregions of the mouse hippocampus with Stereo Investigator.

“Each stereological session started at low magnification with the identification of the boundaries of the region of interest on two sections from each animal,” Dr. Do explained.

Boundaries between the hippocampal regions were traced and an intermediate zone was created between the CA1 and CA3 to ensure these regions did not overlap. The counting and analysis of neurons was performed with the aid of an optical dissector, at a 40x magnification.

“We found Stereo-Investigator user-friendly and adaptable to the requirements of our study, ” said Dr. Do. “We plan to continue using this software to quantify in our animal model the density of neurons in other candidate brain regions implicated in schizophrenia.”

Access the article abstract and full text (by subscription) at jneurosci.org.

Pascal Steullet, Jan-Harry Cabungcal, Anita Kulak, Rudolf Kraftsik, Ying Chen, Timothy P. Dalton, Michel Cuenod, and Kim Q. Do (2010), “Redox Dysregulation Affects the Ventral But Not Dorsal Hippocampus: Impairment of Parvalbumin Neurons, Gamma Oscillations, and Related Behaviors” The Journal of Neuroscience, 30(7):2547-2558

{Image of Dr. Kim Q. Do courtesy of the University of Lausanne}

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Stereo Investigator Aids in Researching Familial Alzheimer’s Disease

Alzheimer’s disease is the most common form of dementia. Most cases occur in people over 65, and are not genetically inherited. Roughly five percent of Alzheimer’s patients suffer from familial Alzheimer’s disease (FAD), an uncommon form that tends to strike sooner, and is related to a genetic predisposition – most commonly, a mutation in the presenilin 1 gene (PS1).

A recent study, led by Dr. Miguel A. Gama Sosa of the Mount Sinai School of Medicine in New York, shows that the mutated PS1 gene contributes to the vascular pathology of familial Alzheimer’s disease.

During the course of their research, Dr. Gama Sosa’s team used Stereo Investigator for the stereologic analyses of transgenic mouse brains.

“We quantified the density, area and length of the vasculature in the hippocampus of mice that express a mutation associated with familial Alzheimer’s disease,” explained coauthor Dr. Gregory A. Elder of the James J. Peters Veterans Affairs Medical Center in the Bronx.

“We found [Stereo Investigator] easy to use and adaptable to the requirements of the study,” he added.

Read “Age-Related Vascular Pathology in Transgenic Mice Expressing Presenilin 1-Associated Familial Alzheimer’s Disease Mutations” at the American Journal of Pathology.

{Image: Anti collagen IV-peroxidase staining of the hippocampal vasculature of a 26 month-old transgenic mouse expressing a human presenilin 1 (PSEN1) transgene harboring the M146V mutation associated with familial Alzheimer’s disease (FAD). Present are vascular abnormalities including string, tortuous and double-barreled vessels.  In addition many vessels are irregular with decreased diameters. – Courtesy of Miguel Gama-Sosa Ph.D.}

Miguel A. Gama Sosa, Rita De Gasperi, Anne B. Rocher, Athena Ching-Jung Wang,  William G.M. Janssen, Tony Flores, Gissel M. Perez, James Schmeidler, Dara L. Dickstein, Patrick R. Hof, and Gregory A. Elder (2010), “Age-Related Vascular Pathology in Transgenic Mice Expressing Presenilin 1-Associated Familial Alzheimer’s Disease Mutations.” American Journal of Pathology, 176:353-368

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