Advanced

Calpain-Mediated Apoptosis in Motor Neurons of Adult Mouse Spinal Cord Slices

Momeni, HamidReza LU (2006)
Abstract
Motor neuron degeneration is a critical phenomenon during spinal cord injuries and some neurodegenerative diseases. However, the mechanisms by which cell death is induced in these neurons are poorly understood. One reason for this, is the lack of model systems in which the survival and death of adult motor neurons can be studied under controlled conditions. This thesis is an attempt to develop such a model system. Here, we tried to establish an in vitro model utilizing slices of adult mouse spinal cord.



A respiratory assay (MTT) was used to assess cell viability in the slices, and cell death was evaluated by morphological features using fluorescent nuclear stains or biochemical feature of cell death using agarose gel... (More)
Motor neuron degeneration is a critical phenomenon during spinal cord injuries and some neurodegenerative diseases. However, the mechanisms by which cell death is induced in these neurons are poorly understood. One reason for this, is the lack of model systems in which the survival and death of adult motor neurons can be studied under controlled conditions. This thesis is an attempt to develop such a model system. Here, we tried to establish an in vitro model utilizing slices of adult mouse spinal cord.



A respiratory assay (MTT) was used to assess cell viability in the slices, and cell death was evaluated by morphological features using fluorescent nuclear stains or biochemical feature of cell death using agarose gel electrophoresis of DNA.



We could demonstrate that both slice thickness and the presence of serum in the culture medium affected slice survival. By using the optimal conditions, we showed that survival could be maintained in the slice cultures for a few days. In the cultured slices, both motor neurons and glial cells died by apoptosis. Interestingly, apoptosis was induced independent of caspase activation, excitotoxicity or free radical formation-classical inducers of apoptosis. Immunohistochemical studies revealed that two isoforms of the Ca2+ -dependent proteases, calpain I and calpain II, appeared in the nuclei of the motor neurons in response to injury and culturing. Calpain activation occurred both in the cytoplasm and the nuclei of the motor neurons as assessed by a fluorogenic calpain substrate. Calpain activation was also observed in the slices by Western blotting using an antibody to 150-kD calpain-cleaved ?-fodrin fragment. Calpain inhibitors and chelation of Ca2+ by EGTA delayed apoptosis and prevented calpain activation in the motor neurons. In contrast, the general caspase inhibitor Z-VAD.fmk had no effect on either apoptosis or calpain activation in the motor neurons.



In conclusion, our results show that massive caspase-independent but calpain-dependent apoptosis is induced in motor neurons in response to injury and culturing and that inhibitors of calpain can delay apoptosis in the motor neurons of cultured slices. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Prof. T├ągerud, Sven, Department of Chemistry and Biomedical Sciences, University of Kalmar, Kalmar, Sweden.
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Calpain, Djurfysiologi, Animal physiology, Spinal cord, Organ culture, Mouse, Motor neurons, Caspase, Calpain inhibitor, Calpain activity, Apoptosis
pages
110 pages
publisher
Department of Cell and Organism Biology, Lund University
defense location
Department of Cell and Organism Biology, Animal Physiology. Building Lecture hall, Helgonav├Ągen 3B, Lund, Sweden
defense date
2006-06-02 09:00
ISBN
91-85067-25-3
language
English
LU publication?
yes
id
856569d2-dc03-48e4-8090-a7482961439b (old id 546762)
date added to LUP
2007-09-06 09:14:26
date last changed
2016-09-19 08:45:08
@phdthesis{856569d2-dc03-48e4-8090-a7482961439b,
  abstract     = {Motor neuron degeneration is a critical phenomenon during spinal cord injuries and some neurodegenerative diseases. However, the mechanisms by which cell death is induced in these neurons are poorly understood. One reason for this, is the lack of model systems in which the survival and death of adult motor neurons can be studied under controlled conditions. This thesis is an attempt to develop such a model system. Here, we tried to establish an in vitro model utilizing slices of adult mouse spinal cord.<br/><br>
<br/><br>
A respiratory assay (MTT) was used to assess cell viability in the slices, and cell death was evaluated by morphological features using fluorescent nuclear stains or biochemical feature of cell death using agarose gel electrophoresis of DNA.<br/><br>
<br/><br>
We could demonstrate that both slice thickness and the presence of serum in the culture medium affected slice survival. By using the optimal conditions, we showed that survival could be maintained in the slice cultures for a few days. In the cultured slices, both motor neurons and glial cells died by apoptosis. Interestingly, apoptosis was induced independent of caspase activation, excitotoxicity or free radical formation-classical inducers of apoptosis. Immunohistochemical studies revealed that two isoforms of the Ca2+ -dependent proteases, calpain I and calpain II, appeared in the nuclei of the motor neurons in response to injury and culturing. Calpain activation occurred both in the cytoplasm and the nuclei of the motor neurons as assessed by a fluorogenic calpain substrate. Calpain activation was also observed in the slices by Western blotting using an antibody to 150-kD calpain-cleaved ?-fodrin fragment. Calpain inhibitors and chelation of Ca2+ by EGTA delayed apoptosis and prevented calpain activation in the motor neurons. In contrast, the general caspase inhibitor Z-VAD.fmk had no effect on either apoptosis or calpain activation in the motor neurons.<br/><br>
<br/><br>
In conclusion, our results show that massive caspase-independent but calpain-dependent apoptosis is induced in motor neurons in response to injury and culturing and that inhibitors of calpain can delay apoptosis in the motor neurons of cultured slices.},
  author       = {Momeni, HamidReza},
  isbn         = {91-85067-25-3},
  keyword      = {Calpain,Djurfysiologi,Animal physiology,Spinal cord,Organ culture,Mouse,Motor neurons,Caspase,Calpain inhibitor,Calpain activity,Apoptosis},
  language     = {eng},
  pages        = {110},
  publisher    = {Department of Cell and Organism Biology, Lund University},
  school       = {Lund University},
  title        = {Calpain-Mediated Apoptosis in Motor Neurons of Adult Mouse Spinal Cord Slices},
  year         = {2006},
}