It is my pleasure to announce details for the 2nd Provence Summer Workshop (Sat 1st September – Tues 4th September 2012), with this year’s theme ‘Molecules & Networks’.

Following on from the great success of our first Workshop, we will once again meet at the beautiful Domaine des Escaunes set in the heart of Provence, France.

We are also delighted to highlight the involvement of our two international guest speakers – Professor Ed Bullmore from Cambridge University (UK) and Professor Mirjana Maletic-Savatic from Baylor Medical College (USA).

This year’s Workshop is certain to be over-subscribed, so register now and take advantage of our early-bird rates. Even better, get involved by presenting your work – submit an abstract for consideration by our scientific committee.

Abstract and Early Bird Registration deadline has been extended to May 31st May 2012 for LAPAD participants as well as anyone attending the Resting State Brain Connectivity conference (Magdeburg, Germany).

See our attached program for more information, as well as the separate registration form which can be sent by fax to +61-2-9351 0551 or email to brainworkshops@gmail.com

I look forward to seeing you in Provence this summer! And feel free to pass this message on to anyone who may be interested.

Regards

Michael Valenzuela

Investigation of the origins of human brain disease has been limited by access to the organ and tissue of interest. The stunning pace of progress in the stem cell field over the last 10 years has overturned this barrier, with multiple possibilities for generating in vitro neurons from self-renewing cell sources. Multipotent neural stem cells (NSCs) isolated from fetal brain extracts were the first reported technique ¹, and soon followed by derivation of pluripotent embryonic stem cells (ESCs) from the inner cell mass of the human blastocyst ². Subsequently, NSCs and neural precursors were generated by transdifferention or redifferentation of somatic stem cell sources, including bone-marrow derived mesenchymal stem cells³ and skin stem cells^4,5. Most recently, there has been intense interest in the production of neurons from adult human fibroblasts through transgenic means – based on either the transduction of pluripotency genes to generate ESC-like induced pluripotential stem cells⁶ (iPS), or transduction of lineage-determining transcription factors to generate neurons directly (induced neuronal cells, iN⁷). The availability of neuronal culture methods based on a readily available patient skin biopsy has therefore heralded a dramatic rise in high-impact studies aimed at in vitro disease modeling of human brain disorders. This includes modeling of inherited brain diseases⁸,  as well as complex neuropsychiatric disorders such as schizophrenia⁹ and Alzheimer’s Disease^10,11.

However, whilst generation of patient-specific neural stem cells (NSC) for the purpose of cell therapy is an exciting new experimental approach for the future treatment of neurodegenerative and neuropsychiatric disorders^9-12, clinical translation is currently limited by two key factors:

1. Efficient generation of sufficient quantities of a homogenous population of NSCs with fate-restricted neuronal differentiation capacity, and
2. Current methods (e.g., human iPS) use of viral vectors to promote aberrant gene expression that is risky for human use¹¹.

Accordingly, we have developed a protocol based on adult canine skin that can efficiently generate homogeneous populations of fate-restricted neural precursors without the use of any genetic manipulation⁵.

The purpose of this study is to adapt and optimise our successful adult canine skin protocol for use with human adult skin, and to comprehensively characterize the resultant cells.

This study will introduce a new technique for producing non-genetically modified neurons from human adult skin. This research may therefore have widespread significant applications in cell therapy, disease cell modeling, drug screening, clinical diagnosis and personalised medicine.

There are two main objectives for this study:

• Develop and optimize a method for the induction, amplification and controlled differentiation of human adult SKiNPs.
• Perform detailed characterisation of these cell lines using a wide range of gene-expression, protein, immunological, morphological, and functional assays

Skin-to-Neurons

 How we obtain our source of stem cells?

Patients undergoing elective orthopedic procedures are invited to participate in the study. After consent is obtained, a donated sliver of skin from the site of surgery is collected and quickly processed in the lab. Stem cells from the tissue sample are isolated and expanded. Afterwhich, the cells are either used to carry out various experiments or frozen down. The frozen cells are used to uphold a stock and build a library of human adult SKiNPs.

From Skin -To – Neurons: Techniques and Methods 

Immunocytochemistry (IHC):

Although antibody staining allows the visualization of neurons in culture, before, during and after the differentiation process, it falls short of showing distinct function of those cells. Therefore, staining is used in combination with other laboratory techniques, to help describe the structure, genetic expression and function of these derived neurons.

Human Adult SKiNPs on matrigel, express classical neural progenitor marker, Nestin (Red) along with the proliferative marker EdU (Green) 

Quantitative PCR: Quite surprisingly, Adult Human SKiNPs express the pluripotent marker Oct-4. This marker is usually expressed in embryonic stem cells, thus indicating to us, that these cells found in the adult, still have genetic characteristics of stemness. 

Electrophysiology: Patch-Clamp

Electrophysiology or patch-clamping will allow us to determine the functional properties of neurons derived from human adult SKiNPs. The patch-clamp method is currently the only technique that permits us to study in-depth the activity of single or multiple channels such as sodium and potassium channels. This is achieved by placing a glass microelectrode onto the membrane of the cell, encapsulating the channels of interest. The membrane is then gently “broken through,” forming a continuous closed circuit, or “whole cell” recording.

References

1          Reynolds, B. A., Tetzlaff, W. & Weiss, S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci12, 4565-4574 (1992).

2          Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science282, 1145-1147 (1998).

3          Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature418, 41-49 (2002).

4          Toma, J. G. et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol3, 778-784 (2001).

5          Valenzuela, M. J., Dean, S. K., Sachdev, P., Tuch, B. E. & Sidhu, K. S. Neural precursors from canine skin: a new direction for testing autologous cell replacement in the brain. Stem Cells Dev17, 1087-1094 (2008).

6          Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell126, 663-676 (2006).

7          Pang, Z. P. et al. Induction of human neuronal cells by defined transcription factors. Nature476, 220-223 (2011).

8          Park, I. H. et al. Disease-specific induced pluripotent stem cells. Cell134, 877-886 (2008).

9          Brennand, K. J. et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature473, 221-225 (2011).

10        Qiang, L. et al. Directed conversion of Alzheimer’s disease patient skin fibroblasts into functional neurons. Cell146, 359-371 (2011).

11        Edelstein, M. L., Abedi, M. R. & Wixon, J. Gene therapy clinical trials worldwide to 2007 – an update. J Gene Med9, 833-842 (2007).

12        Valenzuela, M., Sidhu, K., Dean, S. & Sachdev, P. Neural stem cell therapy for neuropsychiatric disorders. Acta Neuropsychiatr19, 11-26 (2007).

In 2009, she obtained a three-year researching scholarship.  Nowadays, she is completing a PhD in Cognitive Neuroscience under the guidance of Associate Professors Juan García and Lola Roldán-Tapia.  Irene is investigating cognitive reserve, its measurement and its contribution to the delay of cognitive impairment in both clinical and healthy people.  This interesting field drove her to spend two research visits with the Regenerative Neuroscience Group.  Under the supervision of Associate Professor Michael Valenzuela she assisted with the study and analysis of the Lifetime of Experiences Questionnaire based on the two waves of the Memory and Ageing Study.  The link to the RNG continues!  She describes her experience in Sydney as a great enrichment, both professionally and personally.

Listening to music, travelling, learning how to play chess and gazing at artistic creations are some of her favorite pastimes.

Investigation of the origins of human brain disease has been limited by access to the organ and tissue of interest. The stunning pace of progress in the stem cell field over the last 10 years has overturned this barrier, with multiple possibilities for generating in vitro neurons from self-renewing cell sources. Multipotent neural stem cells (NSCs) isolated from fetal brain extracts were the first reported technique ¹, and soon followed by derivation of pluripotent embryonic stem cells (ESCs) from the inner cell mass of the human blastocyst ². Subsequently, NSCs and neural precursors were generated by transdifferention or redifferentation of somatic stem cell sources, including bone-marrow derived mesenchymal stem cells ³and skin stem cells^4,5. Most recently, there has been intense interest in the production of neurons from adult human fibroblasts through transgenic means – based on either the transduction of pluripotency genes to generate ESC-like induced pluripotential stem cells⁶ (iPS), or transduction of lineage-determining transcription factors to generate neurons directly (induced neuronal cells, iN⁷). The availability of neuronal culture methods based on a readily available patient skin biopsy has therefore heralded a dramatic rise in high-impact studies aimed at in vitro disease modeling of human brain disorders. This includes modeling of inherited brain diseases⁸,  as well as complex neuropsychiatric disorders such as schizophrenia ⁹and Alzheimer’s Disease ^10,11.

However, whilst generation of patient-specific neural stem cells (NSC) for the purpose of cell therapy is an exciting new experimental approach for the future treatment of neurodegenerative and neuropsychiatric disorders^9-12, clinical translation is currently limited by two key factors:

1) Efficient generation of sufficient quantities of a homogenous population of NSCs with fate-restricted neuronal differentiation capacity, and

2) Current methods (e.g., human iPS) use of viral vectors to promote aberrant gene expression that is risky for human use¹¹.

Accordingly, we have developed a protocol based on adult canine skin that can efficiently generate homogeneous populations of fate-restricted neural precursors without the use of any genetic manipulation⁵.

The purpose of this study is to adapt and optimise our successful adult canine skin protocol for use with human adult skin, and to comprehensively characterize the resultant cells.

This study will introduce a new technique for producing non-genetically modified neurons from human adult skin. This research may therefore have widespread significant applications in cell therapy, disease cell modeling, drug screening, clinical diagnosis and personalised medicine

There are two (2) main objectives for this study:

• Develop and optimize a method for the induction, amplification and controlled differentiation of human adult SKiNPs.

• Perform detailed characterisation of these cell lines using a wide range of gene-expression, protein, immunological, morphological, and functional assays

Skin-to-Neurons

How we obtain our source of stem cells?

Patients undergoing elective orthopedic procedures are invited to participate in the study. After consent is obtained, a donated sliver of skin from the site of surgery is collected and quickly processed in the lab. Stem cells from the tissue sample are isolated and expanded. Afterwhich, the cells are either used to carry out various experiments or frozen down. The frozen cells are used to uphold a stock and build a library of human adult SKiNPs.

From Skin -To – Neurons: Techniques and Methods 

Immunocytochemistry (IHC):

Although antibody staining allows the visualization of neurons in culture, before, during and after the differentiation process, it falls short of showing distinct function of those cells. Therefore, staining is used in combination with other laboratory techniques, to help describe the structure, genetic expression and function of these derived neurons.

Human Adult SKiNPs on different types of substrata, express classical neural progenitor marker, Nestin (Red) along with the proliferative marker EdU (Green)

CELL PICTURES

Quantitative PCR: Adult Human SKiNPs express early lineage markers of Oct-4, Sox2 and Pax6

GRAPH PICTURE

Electrophysiology: Patch-Clamp

Electrophysiology or patch-clamping will allow us to determine the functional properties of neurons derived from human adult SKiNPs. The patch-clamp method is currently the only technique that permits us to study in-depth the activity of single or multiple channels such as sodium and potassium channels. This is achieved by placing a glass microelectrode onto the membrane of the cell, encapsulating the channels of interest. The membrane is then gently “broken through,” forming a continuous closed circuit, or “whole cell” recording.

PICTURE of Action Potentials and Sodium/Potassium channels goes here

References

1          Reynolds, B. A., Tetzlaff, W. & Weiss, S. A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci12, 4565-4574 (1992).

2          Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science282, 1145-1147 (1998).

3          Jiang, Y. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature418, 41-49 (2002).

4          Toma, J. G. et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol3, 778-784 (2001).

5          Valenzuela, M. J., Dean, S. K., Sachdev, P., Tuch, B. E. & Sidhu, K. S. Neural precursors from canine skin: a new direction for testing autologous cell replacement in the brain. Stem Cells Dev17, 1087-1094 (2008).

6          Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676 (2006).

7          Pang, Z. P. et al. Induction of human neuronal cells by defined transcription factors. Nature476, 220-223 (2011).

8          Park, I. H. et al. Disease-specific induced pluripotent stem cells. Cell134, 877-886 (2008).

9          Brennand, K. J. et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature473, 221-225 (2011).

10        Qiang, L. et al. Directed conversion of Alzheimer’s disease patient skin fibroblasts into functional neurons. Cell146, 359-371 (2011).

11        Edelstein, M. L., Abedi, M. R. & Wixon, J. Gene therapy clinical trials worldwide to 2007 – an update. J Gene Med9, 833-842 (2007).

12        Valenzuela, M., Sidhu, K., Dean, S. & Sachdev, P. Neural stem cell therapy for neuropsychiatric disorders. Acta Neuropsychiatr19, 11-26 (2007).

Hippocampal neurogenesis occurs in several mammalian species, however, the rate of proliferative turnover is less frequent in adult humans compared to rodents. These inter-species differences may reflect intrinsic dorsal versus ventral hippocampal neural precursor differences, and so the canine brain presents an opportunity to test this hypothesis within the one species.

To determine whether there is an increased incidence of neurogenic associated features in the dorsal canine hippocampus compared to the ventral, we employed both histological staining and neural colony forming cell assays.

Newly post-mortem hippocampal canine tissue was isolated, dissociated and suspended in a semi-solid collagen matrix.  By eliminating the possibility of cell fusion, neural precursors could be easily identified and proliferative potential quantified.  Interestingly, a greater number of larger colonies were observed in dorsal hippocampal isolates, thus, the neural colony forming assay is suggestive of increased proliferation rates in the dorsal canine hippocampus (B).  Current efforts are directed at characterising these hippocampal isolates, in attempts to further understand this observed phenomenon.

When working with adult brain tissue, we use histological staining techniques to label cells at different stages of neurogenesis. This allows us to visualise them. Once visualised, we can count them!

In the image below we have used two different histological stains. The first is Cresyl Violet (blue) – this labels for all ‘old’ neurons. The second is doublecortin (brown) – this labels for ‘baby’ neurons, newly formed, and hence is one marker for adult neurogensis.

Read more about RNG research of Canine Neurogenesis as well as an exciting new way of Detecting Neurogenesis in the Living Human Brain.

Rebecca completed a Bachelor of Liberal Studies in 2011, majoring in Anatomy/Neuroscience and Italian Studies. She enrolled in the postgraduate course to explore both the clinical and research paths of neuroscience and is interested in dementia as both a neuroscientific and public health issue.

Otherwise, Rebecca enjoys travelling overseas and learning new languages.  She had an exchange semester at the University of Bologna in Italy, speaks some German, and has studied Spanish in Ecuador.

Our previous research has shown that a more active cognitive lifestyle is not only associated with a reduced risk for long term incident dementia, but also with increased chances of cognitive recovery from Mild Cognitive Impairment to normal cognition. There is growing evidence for the idea that cognitive lifestyle leads to a compression of cognitive morbidity, with important potential socioeconomic implications.

Key Questions

In collaboration with several population-based cohorts around the world, we aim to answer the following key questions:

1. What is a ‘normal’ cognitive lifestyle amongst older Australians?
2. How does cognitive lifestyle compare between older Australians, British, French and Americans?
3. Does a more active cognitive lifestyle help prevent dementia?
4. Which mental activities, if any, are more important?

Status

This study is mid-way through completion. Irene Leon from University of Almeria in Spain has made two visits to RNG to assist in analyzing this data and is currently writing up the results from the Australian LEQ survey.

Outcomes

The following reports related to this study have been published:

• Valenzuela M & Sachdev P. Assessment of Complex Mental Activity Across the Lifespan: Development of the Lifetime of Experiences Questionnaire. Psychological Medicine (2007) 37:1015-1026.
• Valenzuela M, Sachdev P, Chen X, Wen W, Brodaty H. Lifespan mental activity predicts diminished rate of hippocampal atrophy. PLoS One (2008) 3(7):e2598

4Nations Collaborators

• Sydney, Australia – Prof Perminder Sachdev, Sydney Memory & Ageing Study, University of New South Wales
• Cambridge, UK – Prof Carol Brayne & Dr Fiona Matthews, University of Cambridge
• Montpellier, France – Prof Karen Ritchie, University of Montpellier
• New York, USA – Prof Yaakov Stern, Director of Neuropsychology for the Memory Disorders Clinic at the New York State Psychiatric Institute

Funding

This research is funded by the National Health & Medical Research Council (NHMRC).

We conducted a worldwide survey of owners of older dogs (8+ years). We collected over 1000 responses from 11 countries on a 100 different dog breeds and a multitude of cross breeds. From this data we identified the behaviours performed by older dogs that are most indicative of CCD with an accuracy of approximately 80%. These 13 behaviours make up the canine cognitive dysfunction rating (CCDR) scale. Based on this scale, we identified that overall, 12% of the older dogs surveyed had behavioural symptoms consistent with CCD. The risk of having CCD also increased with age with 31% of dogs over the age of 14 years estimated to be affected.

Unfortunately a large proportion of dogs with CCD go undiagnosed as their behavioural changes are dismissed as a normal part of “getting old”. It is hoped that the CCDR scale will make owners and veterinarians more aware of behavioural changes in their older pets and assist in identifying which dogs are “just getting old” from those with CCD.

If you have an older dog and wish to complete the CCDR, it can be found at maturedogs.com. However, it is important to remember that this is only a tool and a consultation with a veterinarian is required to rule out other possible causes of behavioural change.