Progressive neuronal and motor dysfunction in mice overexpressing the serine protease inhibitor protease nexin-1 in postmitotic neurons

Abstract

Perturbation of the homeostasis between proteases and their inhibitors has been associated with lesion-induced or degenerative neuronal changes. Protease nexin-1 (PN-1), a secreted serine protease inhibitor, is constitutively expressed in distinct neuronal cell populations of the adult CNS. In an earlier study we showed that transgenic mice with ectopic or increased expression of PN-1 in postnatal neurons have altered synaptic transmission. Here these mice are used to examine the impact of an extracellular proteolytic imbalance on long-term neuronal function. These mice develop disturbances in motor behavior from 12 weeks on, with some of the histopathological changes described in early stages of human motor neuron disease, and neurogenic muscle atrophy in old age. In addition, sensorimotor integration, measured by epicranial multichannel recording of sensory evoked potentials, is impaired. Our results suggest that axonal dysfunction rather than cell death underlies these phenotypes. In particular, long projecting neurons, namely cortical layer V pyramidal and spinal motor neurons, show an age-dependent vulnerability to PN-1 overexpression. These mice can serve to study early stages of in vivo neuronal dysfunction not yet associated with cell loss.

Fig. 1.

Clinical symptoms in PN-1 overexpressing mice include an age-dependent reduced body weight of TG27 (TG) compared with their wild-type (WT) littermates (A) (*p < 0.05, **p < 0.0001; symbols indicate the mean ± SEM) and a reduced survival rate for TG27 compared with wild-type littermates (B). An analysis of footprint patterns of 6-month-old TG27 (C) and wild-type (D) littermates showed that all parameters related to the stride length were significantly reduced in TG27 mice. LSL, Left stride length; RSL, right stride length;DBF, base of support; l, distance of the right to interposed left hindfoot print; w, width of the stride (female data shown in E; **p< 0.01, *p < 0.05; bars represent the mean ± SD). In addition, the hindlimb extension reflex was abnormal in PN-1 transgenic mice as seen in the pictured 7-month-old TG27 mouse (F) but not in its wild-type littermate (G). Scoring over age shows a significant degree of abnormal reflex in both TG27 (H) and TG24 (I) mice (tightly clasped hindlimbs were scored as 2; p < 0.001, for both lines;symbols indicate the mean ± SEM). See Materials and Methods for number of mice tested.

Fig. 2.

Impaired motor function in PN-1 overexpressing mice was shown in the beam balance performance of TG27 mice (A, p < 0.0001; C, *p < 0.001) but not TG24 (B) mice (a fall was scored as 15 sec) and in the loaded grid test where a transient change was detected in TG27 mice (D), but a significant and lasting deficit was detected in TG24 (E, p < 0.01) mice (symbols indicate the mean ± SEM). See Materials and Methods for number of mice tested.

Fig. 3.

Neurogenic muscle atrophy and axonopathy in PN-1 transgenic mice. A–D, Hematoxylin/eosin-stained (A, B) and NADH histochemically stained (C, D) cross sections of gastrocnemius muscle. Small angular atrophic muscle fibers (arrowhead) intermingled with hypertrophic muscle fibers (arrow), indicating neurogenic muscle atrophy in TG27 (A), are not seen in wild-type (B) littermates. Prominent type I slow (dark) and type II fast (light) fiber clustering indicates chronic denervation and reinnervation in TG27 muscle (C), unlike the normal pattern of randomly scattered fibers from wild-type littermate muscle (D). E–H, p75^NTR immunoreactivity in longitudinal sections of gastrocnemius muscle. Neuromuscular junctions are visualized by α-bungarotoxin binding (E–H,red). Strong anti-p75^NTRstaining (green) is seen at the neuromuscular junctions of 14-week-old TG27 mice (E,H) and 12-week-old TG24 mice (F) compared with wild-type (G) littermates, as well as along nerve fibers (F) and revealed by 2H3 anti-neurofilament staining (H, also red). I,J, Silver-stained longitudinal muscle sections reveal thick, presumably overcompensating nerve fibers along with thin, degenerated ones in some nerve bundles of aged TG27 mice (I) but not wild-type littermates (J). K–N, Transverse sections of lumbar ventral roots. More p75^NTR immunoreactivity (green) is seen in aged TG27 (K) than in wild-type (L) littermates. Neurofilament (SMI-32 antibody) immunoreactivity is similar in TG27 (M) and wild-type (N) littermates. Scale bars:A–D (shown in D), 100 μm; E–H (shown inH), 50 μm; I, J(shown in J), 25 μm;K–N (shown in N), 20 μm.

Fig. 4.

Neuropathological findings in the spinal cord of PN-1 overexpressing mice. A–F, Transverse spinal cord sections of aged littermates stained with an antibody to PN-1 (A–C) or GFAP (D–F). Weak and limited PN-1 cellular immunostaining is seen in wild-type (A) compared with TG27 (B) and TG24 (C) mice; motor neurons are indicated byarrowheads. Far fewer GFAP-positive reactive astrocytes are seen in wild-type (D) than in TG27 (E) and TG24 (F) mice.G, Transient microglial activation in TG27 mice with a peak at 12–14 weeks; insets show isolectin staining of reactive microglia in TG27 but not in wild-type littermates. Bars represent mean ± SEM. H, I, Nonphosphorylated NF-H immunoreactivity in longitudinal spinal cord sections of aged wild-type (H) is strongly reduced in TG27 (I) littermates (ventral funiculus is shown). Scale bars: A–F(shown in F), 200 μm; G,insets, 50 μm; H, I(shown in I), 50 μm.

Fig. 5.

PN-1 and GFAP immunoreactivity in cortex of TG27 and TG24 mice. A–G, Coronal brain sections from the primary motor/somatosensory cortex of littermates stained with an antibody to PN-1 at P20 (A–C) or GFAP at 12 weeks (D–G). Endogenous PN-1 is mainly in layer V pyramidal cells of wild-type mice (A). PN-1 overexpression is strong and regionally restricted in TG27 (B), whereas it is widespread in TG24 (C) mice. Many GFAP-positive reactive astrocytes are found in layers V and VI of TG27 (E; higher magnification in G) but not wild-type (D) or TG24 (F) mice. The cortical layers are indicated by I–VI;asterisk indicates corpus callosum. Scale bars:A–F (shown in F), 200 μm; G, 100 μm.

Fig. 6.

Neuropathological findings in the cortex of TG27 mice. Coronal sections of the hindlimb somatosensory cortex (A, B,E–I) and the primary motor/somatosensory cortex (C, D,J, K) from aged littermates. Hemalum staining of layer V cells shows no difference between wild-type (A) and TG27 (B) mice. Expression of GAP-43 mRNA detected by in situhybridization of wild-type (C) is reduced in TG27 (D) mice. Neurofilament immunoreactivity shows typical somatodendritic localization of nonphosphorylated NF-H in a subset of layer V pyramidal neurons of wild-type mice (E); a strikingly different staining pattern is seen throughout the cortex of TG27 mice (F), where normal (G) as well as abnormal (H) pyramidal neurons, some with aberrant process endings (I), are found. Fluorogold retrograde labeling of layer V pyramidal neurons projecting to the spinal cord in wild-type (J) is reduced in TG27 (K) mice. Scale bars:A, B, E, F(shown in B, F), 100 μm;C, D (shown in D), 100 μm; G–I (shown inI), 12.5 μm; J, K(shown in K), 100 μm.

Fig. 7.

Impairments in motor cortex evoked responses of 6-month-old TG27 mice after whisker stimulation.A, Example of spatiotemporal representation of the cortical responses over the sensory barrel cortex (electrode placed at −2 mm) and the delayed response over the motor cortex (electrode placed at +1 mm); amplitudes (μV) in color; time (ms): horizontal axis; the coordinates related to bregma: up, AP +1; down, AP −3 (mm): vertical axis. B, Quantitative analysis of collective data. Similar amplitudes and latencies are recorded over the sensory barrel cortex of wild-type and TG27 littermates. Significantly decreased amplitude (***p < 0.001) and longer latency (**p < 0.005) are recorded over the contralateral motor cortex of TG27 mice. Bars represent mean ± SEM.