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First published online 3 February 2004
doi: 10.1242/jcs.00940

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The amount of neurofilaments aggregated in the cell body is controlled by their increased sensitivity to trypsin-like proteases

F. Fasani, A. Bocquet, P. Robert, A. Peterson^* and J. Eyer

Laboratoire Neurobiologie and Transgenese, UPRES-EA 3143, INSERM, 4 rue Larrey, bâtiment Montéclair, CHU 49033 Angers CEDEX, France.

View larger version (43K): [in a new window]   Fig. 1 Evaluation of the accumulated amount of neurofilament proteins and transcripts in transgenic and control samples. (A) A typical western blot of brain and spinal cord crude extracts from control (–) and transgenic (+) mice was revealed using antibodies against NFH, NFM and NFL. A much lower amount of each subunit was regularly found in transgenic samples. (B-B') The levels of NFHLacZ mRNA (B') were found to be between 12- and 80-fold lower compared with the endogenous NFH mRNA (B). No major difference in the expression of the endogenous NFH gene was observed between control and transgenic mice during ageing. (C) A 10-fold lower level of NFM mRNA was detected in brain compared with spinal cord without significant difference between control and transgenic tissues. (D-D') A similar pattern of expression of NFL was found between control and transgenic samples. In spinal cord, a 4-fold increase of the light transcript (2.5 kb) has been observed between the earlier stage of life and the other stages.

View larger version (54K): [in a new window]   Fig. 2. Trypsin proteolysis of neurofilaments isolated from control (WT) and NFHLacZ transgenic (TG) mice. (A) Neurofilaments (25 µg) were incubated during 20 minutes with 20 ng (for NFM) or 4ng (for NFH and NFL) of trypsin at 30°C. The reaction was stopped by the addition of Laemmli buffer, boiled during 5 minutes and analyzed by western blot. (B) 50 µg of neurofilaments were digested for 20 minutes at 37°C with trypsin (2 ng for NFH and NFL, and 4 ng for NFM), then sedimented for 30 minutes at 100,000 g and 4°C. Supernatant (S) and pellet (P) fractions were analysed by western blot. Similar proteolytic patterns and solubility of fragments were observed between control and transgenic mice.

View larger version (65K): [in a new window]   Fig. 3. Immunoblot analysis of neurofilaments from control (WT) and NFHLacZ transgenic (TG) mice treated with -chymotrypsin. (A) Isolated neurofilaments (10 µg) were treated during 20 minutes with 4 ng of -chymotrypsin at 30°C, and then analysed by western blotting. (B) Similarly, neurofilaments (50 µg) were digested for 20 minutes at 37°C with -chymotrypsin (2.5 ng for NFH and NFL, or 10 ng for NFM) and centrifuged for 30 minutes at 100,000 g and 4°C. Supernatants (S) and pellets (P) were separated and analysed by western blot.

View larger version (31K): [in a new window]   Fig. 4. Comparison of the extent of neurofilament proteolysis between control and NFHLacZ transgenic samples. Neurofilaments purified from brain were digested at 30°C during increasing times (0-60 minutes) with 4 ng trypsin (A) or -chymotrypsin (B), and the intact subunits were revealed by western blot. Their amount was a proportion of the initial time (t=0). Means and standard deviations of a typical triplicate experiment are shown. Results for control mice are represented by black lanes and those for transgenic NFHLacZ mice by white lanes.

View larger version (76K): [in a new window]   Fig. 5. Distribution of trypsin in the spinal cord of control and NFHLacZ transgenic mice. Frozen spinal cord sections of normal and transgenic mice were immunolabeled with anti-NFH and anti-trypsin antibodies. In transgenic samples a strong immunolocalisation of both epitopes was present in the cell bodies of motor neurons. Merged images are shown on the right.

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