Cell-Specific Loss of SNAP25 from Cortical Projection Neurons Allows Normal Development but Causes Subsequent Neurodegeneration

Abstract

Synaptosomal associated protein 25 kDa (SNAP25) is an essential component of the SNARE complex regulating synaptic vesicle fusion. SNAP25 deficiency has been implicated in a variety of cognitive disorders. We ablated SNAP25 from selected neuronal populations by generating a transgenic mouse (B6-Snap25tm3mcw (Snap25-flox)) with LoxP sites flanking exon5a/5b. In the presence of Cre-recombinase, Snap25-flox is recombined to a truncated transcript. Evoked synaptic vesicle release is severely reduced in Snap25 conditional knockout (cKO) neurons as shown by live cell imaging of synaptic vesicle fusion and whole cell patch clamp recordings in cultured hippocampal neurons. We studied Snap25 cKO in subsets of cortical projection neurons in vivo (L5-Rbp4-Cre; L6-Ntsr1-Cre; L6b-Drd1a-Cre). cKO neurons develop normal axonal projections, but axons are not maintained appropriately, showing signs of swelling, fragmentation and eventually complete absence. Onset and progression of degeneration are dependent on the neuron type, with L5 cells showing the earliest and most severe axonal loss. Ultrastructural examination revealed that cKO neurites contain autophagosome/lysosome-like structures. Markers of inflammation such as Iba1 and lipofuscin are increased only in adult cKO cortex. Snap25 cKO can provide a model to study genetic interactions with environmental influences in several disorders.

Keywords: Drd1a-Cre; Ntsr1-Cre; Rbp4-Cre; SNAP25; neurodegeneration.

Figure 1.

In vivo Cre-expression allows normal development of long-range axonal projections in control and “silenced” Rbp4-Cre expressing cortical L5 projection neurons. Epifluorescent (A, B) and laser scanning confocal microscope tiled images of Rbp4-Cre;Ai14;Snap25^fll+ or Rbp4-Cre;Ai14;Snap25^fl/fl brains. (A, B) images of P2 brains from Rbp4-Cre;Ai14;Snap25^fll+ or Rbp4-Cre;Ai14;Snap25^fl/fl mice, demonstrating presence of tdTom+ cells in the cortex, and tdTom+ fibers projecting subcortically. Boxed region enlarged in (C, D). (C, D) tiled confocal images of tdTom+ fibers in the thalamus and cerebral peduncle (CP) at P2. While projections in the cerebral peduncle are already very abundant and dense, projections to higher order thalamic nuclei such as posterior (Po) nucleus are barely detectable at this age. (C′, D′) tiled confocal images of tdTom+ fibers in the thalamus and cerebral peduncle at P8. By this time point, tdTom+ axons course through dorsal lateral geniculate nucleus (dLGN) and form dense terminal arbourisations in Po and lateral posterior nucleus (LP). The subcerebral projections extend within the cerebral peduncle. The pattern, intensity and fasciculation patterns appear identical in the Rbp4-Cre;Ai14;Snap25^fll+ and Rbp4-Cre;Ai14;Snap25^fl/fl brains. Structures normally devoid of L5 fibers such as habenula do not receive inappropriate innervation in the Rbp4-Cre;Ai14;Snap25^fl/fl brain. Note the absence of tdTomato-expression in the dentate gyrus at P2. Scale bars = 500 μm. Abbreviations: CP, cerebral peduncle; dLGN, dorsal lateral geniculate nucleus; Hb, habenula; LP, lateral posterior nucleus; Po, posterior nucleus; VPM, ventral posterior medial nucleus.

Figure 2.

Cell-type selective knock-down of SNAP25 in Rbp4-Cre;Ai14 layer 5 projection neurons elicited axon disintegration from third postnatal week onwards. Epifluorescent (A, B) and laser scanning confocal microscope tiled images of Rbp4-Cre;Ai14 or Rbp4-Cre;Ai14;Snap25^fl/fl brains. (A) Representative section from a P21 Rbp4-Cre;Ai14 control brain. (B) All projections have developed normally in the Rbp4-Cre;Ai14;Snap25^fl/fl brain by P21, and no cell groups or major axon bundles are missing compared with control brains. Boxes in (A, B) highlight areas of cerebral cortex and dorsolateral geniculate nucleus (dLGN) shown in panels C–F. (C, E) At P21, control and Snap25^fl/fl tdTomato+ cells in the cortex have developed a mature dendrite and abundant local neuropil. (D, F) tdTomato+ L5 neurons in control and Rbp4-Cre;Ai14;Snap25^fl/fl brains have extended axons through dLGN by P21 without forming synapses there. (C′, E′) By P28, large tdTomato+ punctae are visible in the Rbp4-Cre;Ai14;Snap25^fl/fl cortex (arrows in E′) but not in controls. (D′, F′) Axons through dLGN remain continuously labeled at P35. (C″, E″) By P48 the cortex of Rbp4-Cre;Ai14;Snap25^fl/fl brains contains many large tdTomato+ swellings (arrows). (D″, F″) Axons in dLGN are discontinuous (arrowheads) and contain large tdTomato+ swellings in Rbp4-Cre;Ai14;Snap25^fl/fl brains, but remain continuous and without swellings in Rbp4-Cre;Ai14. Scale bars = 500 μm (A, B), 100 μm (C, E, left panel), 50 μm (D, F), or 25 μm (C, E, right panel).

Figure 3.

Rbp4Cre-mediated excision of Snap25^fl results in axon degeneration in the spinal cord. Laser scanning confocal microscope (A–D) or epifluorescent (F) images of Rbp4-Cre;Ai14;Snap25^fl/+ or Rbp4-Cre;Ai14;Snap25^fl/fl brains. (A,A′) at P21, the tdTomato+ axons in longitudinal flat-mounts of the dorsal column of spinal cord (cervical level) from Rbp4-Cre-expressing corticospinal neurons are indistinguishable in Rbp4-Cre;Ai14;Snap25^fl/+ and Rbp4-Cre;Ai14;Snap25^fl/fl mice. (B,B′) By P23, the tdTomato+ axons in the dorsal column show the first signs of large swellings in Rbp4-Cre;Ai14;Snap25^fl/fl mice, but not in controls (C,C′) by the end of the fourth postnatal week, tdTomato+ axons in the dorsal column are reduced in density and the remaining axons show clear signs of discontinuities and axonal swellings in Rbp4-Cre;Ai14;Snap25^fl/fl mice, but axons in control are indistinguishable from those at P21. (D,D′) by P44 no continuous axons are present in the dorsal column of the Rbp4-Cre;Ai14;Snap25^fl/fl mouse, but residual tdTomato+ punctae remain. Control axons remain normal. (E) schematic diagram (Allen Brain Atlas) of the cross-section of the spinal cord at the cervical level, with boxed area indicating the region from which images in panels (F and F′) are taken. (F,F′) at P80 it is evident in cross-sections of the spinal cord, that there are no tdTomato+ axons remaining in the dorsal column of Rbp4-Cre;Ai14;Snap25^fl/fl mice. Scale bars = 100 μm (A–D) and 200 μm (F).

Figure 4.

Rbp4Cre-mediated excision of Snap25^fl results in reduction of VGluT1+ synapses from L5 in posterior nucleus (Po) of the thalamus at P28. Single laser scanning confocal images of tdTomato+ structures and VGluT1+ boutons/punctae in Po thalamus and orthogonal views through the stack of images. (A) VGluT1+ labels glutamatergic presynaptic terminals of cortical axons projecting to Po thalamus. Both of the large tdTomato+ boutons in Po of Rbp4-Cre;Ai14 brains are VGluT1+. (B) VGluT1+ punctae in Po rarely colocalize with tdTomato+ punctae, but one example is shown. (C) Box-and-whisker plot of the quantification of normalized VGluT1+ pixel intensity in tdTomato+ presumed boutons in Po. Note, that some boutons in the cKO are as brightly labeled as those in control brains. Considerably more tdTomato+ boutons in the cKO brains are unlabeled with VGluT1, compared with control brains. Scale bar = 10 μm (applies to both panels).

Figure 5.

Neurodegeneration and inflammation at 8 months of age in “silenced” Rbp4-Cre brains. (A-C′) Tiled laser scanning confocal images of 8mth old Rbp4-Cre;Ai14;Snap25^fl/fl and control brains. Cortical layer boundaries are indicated by short horizontal white bars at the left edge of each panel. (A) L5 Rbp4-Cre;Ai14;Snap25^fl/fl cell with an intact dendrite at 8 months of age. (B, B′) Iba1 immunohistochemistry (green) labels more cells in cKO brains (B′) compared with controls (B). (C, C′) Broadband autofluorescence in cKO and control brains (displayed in green) that probably represents lipofuscin deposits is much more prominent in Rbp4-Cre;Ai14;Snap25^fl/fl (C′) brains compared with controls (C). Inset shows greyscale images acquired across a range of wavelengths (indicated at the top of each panel) of one such autofluorescent particle in an 8-month old brain. (D) Quantification of tdTom+ cell body density in L5 of control and cKO brains at P21, P48 and 8 months of age. A significant reduction in cell body density was only evident at 8 months of age in cKO brains. (E) Quantification of Iba1+ cells below L5 (in the region with most axonal degeneration), within L5 (in the region of cell body death) and above L5. There was a nonsignificant increase in Iba1+ cells in all 3 areas in cKO brains at 8 months of age. (F) The body weight of cKO male and female mice is significantly lower in cKO animals (mean age: 32 weeks cKO females, 31 weeks ctrl females and 27 weeks cKO males and 29 weeks ctrl males). For this graph, only fl/fl (cKO) and fl/+ (ctrl) littermates were analyzed. Scale bar = 100 μm (applies to all panels, except insets). All measures are mean±s.d.; **P < 0.01. Abbreviations: MZ, marginal zone; L2/3, layer 2/3; L4, layer 4; L5, layer 5; L6, layer 6.

Figure 6.

Ultrastructure of neurites in cortex of Rbp4-Cre;Ai14;Snap25^fl/+ or Snap25^fl/fl brains at 8 months of age. (A, B) Examples of tdTomato+ neurites containing autophagosome/lysosome-like vacuoles in Rbp4-Cre;Ai14;Snap25^fl/fl cortex. White arrowheads indicate autophagosome/lysosome-like vacuoles. The boxed regions in A and B are enlarged in A′, A″ and B′, B″, respectively. A′ is an example of multivesicular bodies. A″, B′ and B″ show vacuoles with an electron-dense body. (C) A typical example of tdTomato+ dystrophic neurites in Rbp4-Cre;Ai14;Snap25^fl/fl that contain electron-dense cytoplasm. The boxed region in C is enlarged in C′. The brightness and contrast of C′ were changed to better show immuno-gold labeling, which is indicated by black arrowheads. (D, E) Representative examples of axonal boutons (D) and dendrites (E) in Rbp4-Cre;Ai14;Snap25^fl/+. (F) Representative examples of tdTomato+ axonal boutons in Rbp4-Cre;Ai14;Snap25^fl/fl. The boxed region in F is enlarged in F′ to show multivesicular bodies in the bouton (white arrowheads). (G) Representative examples of tdTomato+ dendrites with dark cytoplasm in Rbp4-Cre;Ai14;Snap25^fl/fl cortex. Abbreviations: Ax, axon; De, dendrite; arrows indicate the boundary of the boutons or dendrites; filled arrowheads point to tdTomato-immunogold label; open arrowheads indicate multivesicular bodies or vacuoles (see legend above); asterisks indicate postsynaptic densities. Scale bars = 500 nm (A, B, D–G) or 1 μm (C).

Figure 7.

Axonal degeneration is present in other cortical Cre-expressing cell populations and shows cell-type specificity in onset and severity. Epifluorescent images of coronal sections of control (Ctrl) and “silenced” (cKO) Rbp4-Cre (A, B), Ntsr1-Cre (D, E) and Drd1a-Cre (G, H) brains, in which Cre-recombinase and tdTomato is expressed in L5, L6 and L6b projection neurons of the cortex respectively. (A′, B′) Tiled laser-scanning confocal images of cortex highlighting the reduction in cell body density in L5 of cKO brain. (A″, B″) Rbp4-Cre;Ai14;Snap25^fl/fl results in absence of axons traversing dLGN at 8 months of age. (C) Quantification of total number of Cre+ L5 neurons in cortex of Ctrl and cKO brains. (D′, D″, D″′) Normal cell density in cortex, intact axons traversing striatum and dense terminal branching in ventrobasal (VB) thalamus of L6 control brains at 8 months of age. (E′, E″, E″′) Ntsr1;Ai14;Snap25^fl/fl results in reduction of cell bodies in cortical layer 6, and absence of striatal axon bundles and terminal synaptic fields in VB of the thalamus that are prominent in the age-matched control brain. (F) Quantification of total number of Cre+ L6 neurons in cortex of Ctrl and cKO brains. (H′, H″, H″′) Drd1a-Cre;Ai14;Snap25^fl/fl brains show little reduction in the sparser axon bundles in striatum (H″) compared with control (G″), but some large punctae are present in cKO striatum (H″). There is a prominent enlargement of punctae in the terminal synaptic field in posterior nucleus (Po) of the thalamus at 8 months of age (H″′) compared with controls (G″′). (I) There is no significant reduction in the number of labeled cell bodies in Drd1a-Cre;Ai14;Snap25^fl/fl brains compared with controls. The time-course of axon degeneration progresses most quickly in L5 axons, at an intermediate pace in L6a axons, but is complete at 8–9 months in both cell populations, but is only beginning in L6b axons by 8 months of age. Images for Rbp4-Cre and Drd1a-Cre are taken at 8 months, images for Ntsr1-Cre are taken at 9–10 months. Scale bars = 1 mm (A, B, D, E, G, H) and 50 μm (all other panels). Abbreviations: Ctx, cortex; dLGN, dorsal lateral geniculate nucleus of the thalamus; Po, posterior nucleus of the thalamus; VB, ventrobasal nucleus of the thalamus.