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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 15, 2005 as doi:10.1096/fj.04-2981fje. |
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,
,1
* Ottawa Health Research Institute, Molecular Medicine Program, Ottawa, Ontario, Canada;
StemPath Inc., Ottawa, Ontario, Canada; and
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada
1 Correspondence: E-mail: lmegeney{at}ohri.ca
SPECIFIC AIM
The present study was designed to examine whether caspase 3 exerted a nonapoptotic role during neural cell differentiation. To this end, we tested the differentiation capacity of neural progenitor cells during peptide inhibition of caspase 3 activity.
PRINCIPAL FINDINGS
1. Caspase 3 activity is elevated during neurosphere differentiation under a nonapoptotic context
Clonally derived neurospheres generated from the striatum of CD1 murine embryos had measurable levels of caspase 3 activity during growth, the activity being localized to single cells within the periphery of the neurosphere. During low serum induction of differentiation (growth factor withdrawal and low serum media), the number of active caspase 3 positive cells surrounding the neurosphere body increased dramatically. Active caspase 3 staining was not readily apparent after 24 h. These results were verified by Western analysis. Western analysis also revealed that PARP remained uncleaved during neurosphere differentiation.
2. Reduction in caspase 3 activity results in decreased expression of neurogenic proteins
To investigate the role of caspase 3 activity during neurogenesis, CD1 neurospheres were incubated in differentiation media supplemented with either the caspase 3 inhibitor z-DEVD-fmk or DMSO as a control. During growth conditions, both control (DMSO panels) and caspase 3-inhibited neurospheres (DEVD panels) demonstrated high levels of nestin (Fig. 1
A), a marker of early neural progenitor cells. After 48 h in differentiation media, control neurospheres had low levels of nestin protein (Fig. 1B
). These cells also expressed markers characteristic of astrocytes and oligodendrocytes as indicated by GFAP (Fig. 1C
) and MBP (Fig. 1D
) immunocytochemistry, respectively. Conversely, neurospheres incubated in differentiation media in the presence of z-DEVD-fmk retained nestin expression after 48 h (Fig. 1B
) and did not readily stain for either GFAP (Fig. 1C
) or MBP (Fig. 1D
). Caspase 3-inhibited neurospheres displayed very low levels of the neuronal differentiation marker ß-III tubulin. We also noted a similar differentiation deficit in neurospheres that were derived from caspase 3 null embryos.
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3. Inhibition of caspase 3 activity attenuates neurite extension and cellular remodeling during differentiation
Dendritic extension and axonal branching is a key feature of neurogenesis. This phenotype was not apparent in neurospheres incubated with caspase 3 inhibitors during differentiation. Neurospheres incubated with a caspase 8 inhibitor did not have adverse effects on neurite extension whereas the use of a pan-caspase inhibitor gave results similar to those observed with caspase 3 inhibition alone. These results suggest that in addition to enhancing the expression of a postmitotic gene expression program, caspase 3 activity also mediates the morphologic adaptations that accompany cellular differentiation.
4. The activity of proneurogenic kinases are reduced with caspase 3 inhibition
To investigate a possible mechanism for caspase 3 induction of neurogenesis, we focused our efforts on identifying the relevant caspase 3 substrates that would relay the differentiation signal(s). Caspase proteases have been shown to initiate apoptotic signaling pathways through cleavage activation of protein kinases. As such, the lack of neurite extensions with caspase 3 inhibition directed us to examine the activity of protein kinases that regulate cellular remodeling. The PAK kinase family was especially interesting as PAKs are known to be regulators of cytoskeletal dynamics and cell motility. We observed a striking reduction in PAK1 activity when neuroblasts were incubated with a caspase 3 inhibitor peptide (Fig. 2
A, B). ASK1 is a kinase that has been associated with both apoptosis and differentiation. Similar to PAK1, caspase 3 inhibition reduced the activity of ASK1 during neuroblast differentiation (Fig. 2C, D
). The p38
MAPK is targeted by both PAK1 and ASK1. We found a dramatic reduction in p38 activity in caspase 3 inhibited neuroblasts (Fig. 2E, F
), suggesting that PAK/ASK induction of p38 activity is a conserved feature of neuronal stem cell differentiation.
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CONCLUSIONS AND SIGNIFICANCE
This study demonstrates a nonapoptotic role for caspase 3 in differentiating neural progenitor cells. We noted that caspase 3 activity was elevated during neurosphere differentiation in the absence of apoptosis. Using a peptide inhibitor of caspase 3, we observed a dramatic attenuation in the expression of neurogenic proteins specific for the formation of neurons, astrocytes, and oligodendrocytes. These findings were also supported by the observation that neurospheres cultured ex vivo from caspase 3 null mice displayed similar differentiation deficits. We also observed that caspase 3-inhibited neurospheres had strikingly fewer neurite extensions than did control neurospheres when incubated in differentiation media. We determined that caspase 3 inhibition resulted in attenuated activities of PAK1, ASK1, and p38. These kinases have been implicated in both apoptosis and differentiation. Based on these observations, it is likely that caspase 3 activation in neural cell populations is not exclusive for apoptotic outcomes.
Evidence describing nonapoptotic functions for caspase proteases have recently been reported in a variety of model systems. Caspase proteases are now known to promote sperm cell maturation, as well as the differentiation of keratinocytes, erythroids, monocytes, myoblasts, and lens fiber cells. Clearly, non-death-related caspase activity is not limited to a particular cell or subset of cells. Therefore, the factors that determine the outcome of caspase activation (i.e., apoptotic or nonapoptotic) are likely present in many different cell types and depend upon the cellular environment (Fig. 3
).
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The targeted activation of signaling proteins by caspases has emerged as a common theme in studies describing the effects of cellular caspase activity. In many instances, the kinases affected by caspase activity also mediate signals that provoke the phenotypic changes associated with cellular differentiation/transformation. Enzymes such as MEK kinase 1 (MEKK1), ste20-like kinase (SLK), and the p21-activated kinases have important roles in cellular remodeling and are known to be cleavage-activated by caspase 3. In the cellular context investigated, these kinases were reported to promote apoptosis. Nonetheless, it is conceivable that caspase-directed activation of these kinases may also have nonapoptotic outcomes when activated under a different circumstance. Factors such as caspase-substrate and kinase-substrate stoichiometry, strength, and duration of caspase activity may determine the cellular outcome mediated by this protease family (Fig. 3)
.
Antiapoptotic therapies designed to limit the activity of caspase proteases have been described as effective strategies to reduce neurodegeneration in conditions such as Parkinson’s disease, Alzheimer’s disease, and ischemia-induced brain injury. These studies have demonstrated effective limitation of apoptosis using caspase inhibitory proteins such as neuronal apoptosis inhibitory protein (NAIP), and X chromosome-linked inhibitor of apoptosis (XIAP). Peptide inhibition of caspase activity was also reported to successfully attenuate apoptosis. However, the validity of such therapeutic approaches has been challenged by studies that report confounding results (i.e., these caspase inhibitory proteins promoted an unanticipated disruption in ambient neuron function). The results from our study describing a nonapoptotic role for caspase proteases in neuronal differentiation strengthen this argument.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2981fje;
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