Lab Overview
The Miles laboratory is investigating, at the levels of cellular receptors
and gene expression, the regulation of the activity of plasmin, a key protease
that regulates pathophysiological processes involving extracellular matrix
degradation, tissue remodeling, cell migration, and apoptosis.
Highlight
Role of the Plasminogen Activation System in Hippocampal Neuritogenesis
Hongdong Bai, Leif Dehmelt, Shelley Halpain, Victoria A. Ploplis, Francis J.
Castellino, Robert J. Parmer, Lindsey A. Miles
A major role for the plasminogen activation system in neuronal function has
been established in the literature. Here we report that the plasminogen activation
system regulates neuritogenesis of hippocampal neurons. When 0.5 µM plasminogen
was added to primary cultures of murine hippocampal neurons, the number of
neurite positive cells after 24 hr of culture increased from 35.37 ± 2.2%
to 61.09 ± 3.8% (p<0.05) compared to untreated neurons. After 72
hr of culture, the average neurite length increased from 68.34 ± 0.6
µm to 110.3 ± 1.4µm (p<0.001) by plasminogen treatment. Similar
results were obtained with embryonic rat hippocampal neurons. Furthermore,
differential immunostaining of rat hippocampal neurons indicated that the average
axon length increased from 51.8± 6.24 µm to 106.6 ± 6.4 µm (p<0.01)
and the average dendrite length was increased from 42.33 ± 4.5 µm to
87.7 ± 5.6 µm (p<0.01). Conversely, the growth cone areas of the
hippocampal neurons were markedly decreased following plasminogen treatment,
consistent with the idea that larger growth cones are often stationary and
contribute less to neurite outgrowth. The stimulation of neuritogenesis by
plasminogen was abolished in the presence of aprotinin, suggesting that the
activation of plasminogen by endogenous plasminogen activators was required.
Therefore, we examined neurite outgrowth in t-PA -/- mice. Neurite length and
the number of neurite positive cells were not statistically different when
comparing t-PA -/- vs wild type hippocampal neurons. However, application of
amiloride, an inhibitor of u-PA, decreased neurite outgrowth from wild type
hippocampal neurons [69.8 ± 2.5 µm vs 43.28 ± 2.5 µm (p<0.05)
for untreated vs amiloride-treated, respectively]. Interestingly, the inhibitory
effect of amiloride on the length of t-PA -/- neurons (64.33 ± 3.6 µm
vs 33.67 ± 1.4 µm for untreated t-PA -/- cells vs amiloride-treated
t-PA -/- cells, respectively] was significantly greater (p<0.005) than the
effect on wild type neurons. There was a statistically significant difference
between amiloride treated wild type (43.28 ± 2.5 µm) and amiloride treated
t-PA -/- neurons (33.67 ± 1.4 µm) (p<0.05). These results suggest
that both neuronal u-PA and neuronal t-PA participate in hippocampal neurite
outgrowth. In addition, a direct interaction of plasminogen with the hippocampal
neurons appeared to be required because µ-aminocaproic acid (EACA) abolished
the enhancement of neurite outgrowth by mouse hippocampal neurons following
the addition of plasminogen [50.72 ± 3.8 µm vs 139.4 ± 6.3 µm
(p<0.005) for EACA + plasminogen vs plasminogen alone, respectively]. EACA
also decreased neurite positive cells in untreated hippocampal neurons [86.66 ± 5.9%
vs 68.75 ± 4.5% (p<0.05) for untreated vs EACA treated, respectively],
suggesting that endogenous neuronal plasminogen is required for optimal neuritogenesis.
Accordingly, we found that neurite outgrowth was significantly decreased in
plasminogen deficient hippocampal neurons, compared to wild type controls [43.38 ± 1.1
µm vs 62.18 ± 2.25 µm (p<0.02) for plasminogen deficient vs wild
type hippocampal neurons, respectively]. These observations suggest that endogenous
plasminogen is required for optimal hippocampal neuritogenesis. Furthermore,
active plasmin is required. Finally, sufficient plasminogen activation can
be accomplished by either endogenous t-PA or endogenous u-PA.
2006 Publications
Mitchell, J.W., Baik, N. Castellino, F. J., Miles, L. A. Plasminogen inhibits
TNF? apoptosis in monocytes, Blood, 107(11):4383-4390, 2006.
Miles, L. A., Andronicos, N. M., Baik, N., Parmer, R. J. Cell surface actin
binds plasminogen and modulates neurotransmitter release from catecholaminergic
cells, J. Neurosci., 26: 13017-13024, 2006.
