Main Text

Synapse elimination is the process in which excess synapses formed early during brain development are subsequently eliminated to form mature circuits (1–3). The outcome of synapse elimination depends on both spontaneous and experience-driven neural activity, with less active synapses being preferentially removed (2, 4, 5). However, the molecular mechanisms that connect neuronal activity to synapse elimination remain unclear. Previous studies have proposed that microglia and astrocytes, two major glial cell types of the brain, mediate activity-dependent synapse elimination by engulfing weak synapses (6–14), but it is unclear how glia can detect the “strength” of a synapse. A recent study proposed a molecular mechanism for activity-dependent synapse elimination in which the JAK2-STAT1 pathway is activated in inactive callosal projection neurons and regulates the removal of synapses and axons (15). However, the JAK-STAT pathway canonically functions through transcriptional regulation (16), which affects entire cells. If not all synapses made by a neuron are destined for removal (3), activation of the JAK2-STAT1 pathway alone may not provide sufficient elimination specificity. Therefore, other unidentified, locally-effective mechanisms likely exist that confer elimination specificity at the synapse level.

Caspase-3 is a protease crucial for the execution of apoptosis (17). In hippocampal neurons, transient activation of caspase-3 is required for long-term depression (LTD), a form of synaptic plasticity that induces long-lasting decreases in synaptic strength (18). Caspase-3 activation in apoptotic cells is also known to lead to the clearance of these cells by phagocytes (19, 20). These findings led us to hypothesize that caspase-3 may link synapse weakening with synapse removal by glia. Although caspases were previously implicated in dendrite pruning in metamorphosis, degeneration-like axon elimination, and the regulation of hippocampal spine density (21–24), the role of caspase-3 in synapse elimination in response to neural activity was not clear. In this work, we identify caspase-3 as a key molecule that is activated in postnatal development in dendritic compartments upon synaptic weakening and is necessary for elimination of weak synapses by microglia. Furthermore, we discovered that caspase-3 deficiency protects against amyloid-β-induced synapse loss in a mouse model of Alzheimer’s disease, highlighting its significance not only in developmental synapse elimination but also in adult neurodegenerative diseases.

Tetanus toxin expression inactivates retinogeniculate synapses

We chose the mouse retinogeniculate visual pathway as a model system to study synapse elimination (25). In mice, retinal ganglion cells (RGCs) in each eye send out axons to form retinogeniculate synapses with relay neurons in the dorsal lateral geniculate nuclei (dLGN) of the thalamus on both sides of the brain (Fig. S1) (25). The majority of RGC axons from each eye innervate the contralateral (on the side opposite to the originating eye) dLGN while a smaller fraction innervate the ipsilateral (on the same side as the originating eye) dLGN (Fig. S1) (25). Regions in each dLGN innervated by the two eyes initially overlap but later segregate into eye-specific territories (Fig. S1). This segregation process is a hallmark of retinogeniculate pathway development at the morphological level and depends on synapse elimination and spontaneous RGC activity (25–27).

To investigate the role of caspase-3 in activity-dependent synapse elimination, we first needed to establish a method that could manipulate the strength of a subset of retinogeniculate synapses. For this purpose, we used adeno-associated-virus (AAV) to deliver a construct expressing tetanus toxin light chain (TeTxLC) under the control of the neuron-specific human synapsin promoter (AAV-hSyn-TeTxLC) (Fig. 1A) (28). In utero intraocular injection of AAV-hSyn-TeTxLC at embryonic day 15 (E15) leads to TeTxLC expression in RGCs by the time of birth, blocking neurotransmitter release at retinogeniculate synapses by cleaving synaptobrevin and leading to synapse inactivation (28, 29). To label RGC axons and facilitate quantification, we co-injected AAVs that express fluorescent proteins (mTurquiose2, eGFP, or tdTomato, depending on the experiment) as anterograde tracers (Fig. 1A). These AAVs expressing fluorescent proteins also served as controls for eye injections.

Figure 1.

To validate the efficacy of our synapse inactivation method, we injected AAV-hSyn-TeTxLC into the right eye of wildtype E15 embryos and analyzed the segregation of eye-specific territories at postnatal day 8 (P8), when the segregation process is largely complete (25). To quantify the overlap between eye-specific territories in an unbiased manner, we applied a set of increasing thresholds to signals from both eyes (Fig. S2) (30). For each threshold, we calculated a percentage overlap value as the ratio of the dLGN area with signals from both eyes to the total dLGN area (Fig. S2 and Fig. S3). Consistent with previous results (26, 27), inactivating right eye-associated retinogeniculate synapses led to contraction of right eye-specific territories and expansion of left eye-specific territories relative to control animals (Fig. S3A and S3B). Importantly, segregation of eye-specific territories in TeTxLC-injected mice was significantly defective (Fig. S3C and S3D), confirming that our method potently inactivated synapses and perturbed activity-dependent refinement of the retinogeniculate pathway.

Synapse inactivation induces postsynaptic caspase-3 activity

Is caspase-3 activated in response to synapse weakening? To address this question, we injected AAV-hSyn-TeTxLC into the right eyes of E15 embryos and looked for caspase-3 activation in dLGNs of TeTxLC-injected mice at the age of P5 (a time when synapse elimination is most active) (Fig. 1A) (7, 25). We used immunohistochemistry against the cleaved and active form of caspase-3 to detect caspase-3 activity in the dLGN area (17). If synapse inactivation leads to caspase-3 activation, we would expect higher levels of caspase-3 activity in dLGNs with more inactivated synapses (Fig. 1A). In agreement with this hypothesis, caspase-3 activity in left dLGNs of TeTxLC-injected animals (dLGNs with the most inactivated synapses) was significantly higher compared to that in right dLGNs of the same animals (dLGNs with intermediate amount of inactivated synapses) (Fig. 1B, 1C, and 1E) and compared to that in dLGNs of animals receiving control injections in the right eyes (dLGNs with no inactivation) (Fig. 1B, 1D, and 1E). Reassuringly, caspase-3 activities in left and right dLGNs of control animals were comparable (Fig. 1E), despite injections being administered only in the right eyes. This suggests that surgical manipulation and viral transduction had no measurable contribution to caspase-3 activation. These findings support the model that inactivation of retinogeniculate synapses leads to caspase-3 activation.

Does inactivation of retinogeniculate synapses activate caspase-3 pre-synaptically in RGCs or post-synaptically in dLGN relay neurons? Caspase-3 activity in dLGNs of TeTxLC-injected mice was present either as discrete, punctate signals (Fig. 1F) or as staining of entire cells (Fig. 1B and Fig. S4Bi-iii). Both types of signals were more abundant in dLGNs with strong synapse inactivation than in control dLGNs (Fig. 1B, 1D and 1F). Intriguingly, punctate caspase-3 activity was not found within TeTxLC-expressing RGC axons (Fig. 1G). Instead, punctate caspase-3 activity closely juxtaposed inactivated axon terminals (Fig. 1G) and co-localized with the dendritic marker MAP2 (Fig. S4A). In the case where caspase-3 was activated in entire cells, the cells were positive for the neuronal marker NeuN (Fig. S4C) and possessed relatively large round somas and multipolar dendritic arbors (Fig. S4Bi-iii), all of which are characteristics of dLGN relay neurons (31, 32). These observations suggest that caspase-3 activation triggered by synapse inactivation occurs predominantly in the dendritic compartments of dLGN relay neurons that are postsynaptic to the inactivated synapses.

Although morphologically distinct, localized and whole-cell caspase-3 activity may be mechanistically linked. In support of this, we observed transitional states of caspase-3 activation where multiple dendrites of a relay neuron were positive for active caspase-3 without the cell body becoming apoptotic (an example is shown in Fig. S4Biv), suggesting that whole-cell caspase-3 activation may arise from accumulation of local caspase-3 activity. Such transitions might be particularly relevant in dLGNs that receive the majority of TeTxLC-expressing axons, as systematic silencing of synapses could lead to widespread caspase-3 activation in the postsynaptic neuron, potentially overwhelming negative regulatory mechanisms that normally keep caspase-3 activation localized (24, 33–35). While our data cannot determine which form of caspase-3 activation plays a more significant role in the normal development of the visual pathway, the infrequent occurrence of whole-cell caspase-3 activation in control dLGNs (Figure 1D) -- which undergo normal synapse elimination -- suggests that localized caspase-3 activation is necessary to drive synapse elimination under physiological conditions.

Caspase-3 activation at weak synapses requires the presence of strong synapses

Previous experiments have manipulated RGC neuronal activities and demonstrated that, regardless of the absolute level of RGC activity, the dLGN territory of less active RGCs always contracted relative to that of more active RGCs (26, 27). This observation suggests that the outcome of synapse elimination is driven by competitive interactions between strong and weak synapses. Consistent with this idea, a recent study demonstrated that elimination of weak synapses in callosal projections occurred only when other strong synapses are present (28, 36).

To test if caspase-3 activation at weak synapses requires the presence of strong synapses, we designed two experimental conditions. In the first condition, we inactivated RGCs only in the right eye, leading to the presence of both active (from the left eye) and inactive (from the right eye) synapses in the dLGN (Fig. 2A). We termed this condition “single inactivation”. In the second condition, we inactivated RGCs in both eyes, leading to the presence of only inactive synapses in the dLGN (Fig. 2A). We termed this condition “dual inactivation”. We also included a control condition where no RGCs were inactivated (Fig. 2A). We first confirmed that, in accordance with previous findings (26, 37), segregation of eye-specific territories was disrupted in the dual inactivation condition (Fig. S5A-D). We then measured dLGN caspase-3 activity in control, single, and dual inactivation conditions. Intriguingly, while dLGN caspase-3 activity was significantly higher in the single inactivation condition compared to that in the control (Fig. 2B and 2C), dLGN caspase-3 activity in the dual inactivation condition was not significantly different from that in the control (Fig. 2B and 2C) and was significantly lower than that in the single inactivation condition (Fig. 2B and 2C). Although not reaching statistical significance, dLGN caspase-3 activity on average was still higher in the dual inactivation condition compared to the control (Fig. 2C), possibly due to incomplete inactivation of retinogeniculate synapses, or interaction between weak retinogeniculate synapses and strong synapses from other pathways, or additional mechanisms that activate caspase-3 independently of interactions between weak and strong synapses. These results suggest that interaction between weak and strong synapses is required for caspase-3 activation to occur at weak synapses.

Figure 2.

Caspase-3 is required for segregation of eye-specific territories

Is synapse inactivation-induced caspase-3 activity important for synapse elimination in the retinogeniculate pathway? To address this question, we investigated whether segregation of eye-specific dLGN territories is defective if caspase-3 activation does not occur. Since we were unable to find a Cre-expressing mouse line that satisfactorily depletes caspase-3 in dLGN relay neurons, we used full-body caspase-3 deficient (Casp3^−/−) mice (38, 39). These mice were backcrossed to congenicity on the C57Bl/6J background and were viable, fertile, and did not exhibit overt abnormalities (39). To measure segregation of eye-specific territories, we injected the eyes of Casp3^+/+ and Casp3^−/− littermate mice at P9 with fluorophore-conjugated cholera toxin subunit B (CTB), using a different fluorophore for each eye. CTB molecules bind to ganglioside molecules on RGC surfaces and anterogradely label RGC axon terminals (40). At P10, when CTB labeling and eye-specific segregation were complete (25, 30), we collected the brains and imaged eye-specific territories in each dLGN. By visual inspection, eye-specific territories in Casp3^+/+ dLGNs overlapped minimally (Fig. 3A). In contrast, Casp3^−/− dLGNs showed clear defects in the separation of eye-specific territories (Fig. 3B) (41). We then quantified eye-specific segregation as described previously (Figure S2) and found that percentage overlap in Casp3^−/− dLGNs was consistently higher than that in Casp3^+/+ dLGNs across all thresholds (Fig. 3C and D). Importantly, the fold difference and statistical significance of the difference in percentage overlap between the two groups of animals increased as the cutoff threshold increased (Fig. 3D), suggesting the defects observed in Casp3^−/− animals cannot be attributed to artifacts generated from background noise.

Figure 3.

As RGCs are known to undergo extensive apoptosis during the first week of postnatal development (42, 43), we wondered if caspase-3 deficiency might block RGC turnover and result in more RGC inputs in the dLGN, thereby confounding the overlap analysis. To address this concern, we quantified the density of RGCs in whole-mount retinae using a RGC marker, RBPMS (Fig. S6A) (44). Reassuringly, RGC densities in retinae of Casp3^+/+ and Casp3^−/− mice were not different (Fig. S6B), indicating that RGC apoptosis still occurred at normal rates in the absence of caspase-3, presumably through redundant mechanisms.

It is also possible that caspase-3 deficiency might confound the overlap analysis by affecting the number of dLGN relay neurons. Therefore, we used NeuN staining to visualized relay neurons in dLGNs of Casp3^+/+ and Casp3^−/− mice and confirmed that relay neuron density is not significantly altered by caspase-3 deficiency (Figure S7). Notably, this observation indicates that, during normal development, synapse weakening does not induce widespread whole-cell caspase-3 activation in dLGN relay neurons, corroborating the idea that localized caspase-3 activity drives synapse elimination in dLGN. Collectively, our findings suggest that caspase-3 is required for normal segregation of eye-specific territories in dLGN.

Caspase-3 is required for retinogeniculate circuit refinement

Segregation of eye-specific territories in the mouse dLGN occurs prior to eye opening (which happens at ∼P13) and relies predominantly on spontaneous RGC activity (25). After eye opening, sensory-dependent RGC activity drives a second phase of refinement at the circuit level (25). At the time of eye opening, each dLGN relay neuron receives numerous weak RGC inputs. With visual experience, a minority of these inputs are strengthened while other weak inputs are eliminated, so that by P30 only a few (typically ≤3) strong RGC inputs innervate each dLGN relay neuron (25).

To test if caspase-3 is required for visual experience-driven circuit refinement, we prepared acute brain slices from P30 Casp3^+/+ and Casp3^−/− littermate mice and investigated the electrophysiological properties of retinogeniculate synapses (Fig. S8A) (45, 46). We stimulated the optic tract while recording both AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated excitatory postsynaptic currents (EPSCs) in dLGN relay neurons (Fig. S8A and Fig. 4A-B). By gradually increasing the stimulation intensity on the optic tract, we recruit individual RGC axons in a serial manner (Fig. S8A). Excitation of each RGC axon triggers a step increase in the EPSC amplitude (Fig. S8A), allowing us to infer RGC input numbers by counting steps in EPSC response curves (Fig. 4C and S9A). In agreement with previous studies, retinogeniculate circuits in Casp3^+/+ animals were refined, with dLGN relay neurons most frequently receiving 2 strong RGC inputs, and the great majority of neurons receiving no more than 3 inputs (Fig. 4A, 4C and S9A). In contrast, RGC input counts of dLGN relay neurons in Casp3^−/− animals followed a long-tailed distribution that was significantly right-shifted from that of Casp3^+/+ animals (Fig. 4C and S9A). The majority of Casp3^−/− relay neurons received more than 3 inputs (Fig. 4B-C and S9A), and many inputs elicited only small increments in EPSC amplitude (Fig. 4B). Additionally, we examined maximum EPSC amplitude (Fig. S9B-C), AMPAR-EPSC to NMDAR-EPSC ratio (Fig. S9D), and fiber fraction (Fig. S9E) (46) in Casp3^+/+ and Casp3^−/− mice. We found no significant difference between the two groups of animals, suggesting that the defective circuit refinement in Casp3^−/− animals was not due to the lack of retinogeniculate synapse maturation but instead an inability to eliminate weak synapses.

Figure 4.

An increased number of RGC inputs innervating dLGN relay neurons in Casp3^−/− mice should correspond to greater numbers of presynaptic release sites and should result in more frequent spontaneous release of neurotransmitters. To test this prediction, we measured miniature EPSCs (mEPSCs) in dLGN relay neurons of Casp3^+/+ and Casp3^−/− mice (Fig. 4D). We found that mEPSCs in Casp3^−/− neurons occurred more frequently (Fig. 4E), with a greater fraction of them having small amplitudes (Fig. 4F). These results mirror previous measurements in hippocampal CA1 neurons in Casp3^−/− mice (24). Overall, our observations are consistent with the model where caspase-3 deficiency impairs the removal of weak synapses, leading to greater number of inputs on dLGN relay neurons.

As an additional way to probe retinogeniculate circuit refinement, we measured paired-pulse response ratios (PPRs) in dLGN relay neurons (Fig. S8B and Fig. 4G-H) (45, 47). The retinogeniculate circuit typically generates PPRs that are smaller than one due to depletion of neurotransmitters (Fig. S8B) (45, 47). If there is an increase in the number of release sites in Casp3^−/− mice, we expect more available neurotransmitters and larger PPRs (Fig. S8C) (47). Indeed, we observed higher PPRs in Casp3^−/− dLGN relay neurons (Fig. 4D-E), suggesting that defective synapse elimination resulted in more RGC release sites in Casp3^−/− mice. Taken together, our results demonstrate that caspase-3 is required for experience-driven retinogeniculate circuit refinement.

Caspase-3 deficiency impairs synapse elimination by microglia

Our results demonstrate that proper synapse elimination in the retinogeniculate pathway requires caspase-3 activity. Previous studies have shown that microglia and astrocytes engulf weak synapses during the process of synapse elimination (7, 10). We therefore sought to determine whether caspase-3 activation is required for synapse elimination by microglia and/or astrocytes.

If synapse elimination by microglia depends on caspase-3 activation, then microglia in Casp3^−/− mice should engulf less synaptic material. Similar to a previous study (7), we utilized the Cx3cr1-Gfp transgenic mouse line to visualize microglia in vivo. In this line, the native Cx3cr1 promoter drives Gfp expression and specifically labels microglia in the brain (Fig. 5A) (48). We labeled RGC axon terminals by injecting fluorophore-conjugated CTB in the eyes of P4 Casp3^+/−; Cx3cr1-Gfp^+/− and Casp3^−/−; Cx3cr1-Gfp^+/− littermate pups (Casp3^+/− instead of Casp3^+/+ mice were used to enable more efficient breeding) (7). At P5, the brains were harvested and the dLGN imaged (Figure 5A). Microglia in Casp3^−/−; Cx3cr1-Gfp^+/− mice had small soma and thin processes, indicating that caspase-3 deficiency did not result in microglial activation (Fig. 5B). This is further validated by immunohistochemical analysis of microglia in caspase-3 deficient mice (Fig. S10A-D). To quantify synapse engulfment, we isolated volumes corresponding to microglia and RGC axon terminals in the dLGN (Fig. 5B) and calculated the volume of RGC terminals that were fully enclosed in each microglia (Fig. 5C). As expected, we observed that microglia in Casp3^−/−; Cx3cr1-Gfp^+/− mice engulfed significantly less axonal material compared to those of Casp3^+/−; Cx3cr1-Gfp^+/− littermates (Fig. 5B-C). The same trend was observed when RGC axon terminals originating from the two eyes were analyzed separately (Fig. S11A-B). As the engulfment capacity of microglia is limited by their size, we normalized the volume of engulfed synaptic material in each microglia to the microglial volume (Fig. 5D). Microglia in both groups of mice are comparable in size (Fig. S11C), and the defective synaptic engulfment in Casp3^−/− mice persisted after normalization to microglial volume (Fig. 5D). Additionally, the same defect was observed when engulfment values were averaged and reported by animal (Fig. S11D). Taken together, our results demonstrate that microglia-mediated synapse elimination depends on caspase-3.

Figure 5.

To test whether caspase-3 activation is also required for astrocyte-mediated synapse elimination, we used the Aldh1l1-Gfp transgenic mouse line, where Gfp expression specifically labels astrocytes (Fig. S12A) (49). We again labeled RGC axon terminals in Casp3^+/−; Aldh1l1-Gfp^+/− and Casp3^−/−; Aldh1l1-Gfp^+/− littermate mice with CTB conjugates (Fig. S12A). Astrocytes were present in the P5 dLGN at a much higher density than microglia and possess very fine processes (Fig. S12A). To ensure segmentation fidelity, we only analyzed astrocyte cell bodies and their proximal processes (Fig. S12B). Unexpectedly, we found that the volume of axonal material engulfed by astrocytes was comparable in the two groups of mice (Fig. S12C). The same trend was observed when we normalized the volume of engulfed synaptic material to astrocyte volume (Fig. S12D-E). It is possible that, by using Casp3^+/− mice instead of Casp3^+/+ mice as controls, we have obscured some defects in astrocyte-mediated synapse elimination. Nevertheless, given that astrocyte-mediated synapse elimination did not show clear dependence on caspase-3, we focused on measuring synapse engulfment by microglia as a readout of preferential elimination of weak synapses.

Removal of weak synapses by microglia requires caspase-3 activity

If caspase-3 activation at weak synapses is important for synapse removal, then in caspase-3 deficient mice, microglia should lose the ability to engulf weak synapses. To test this hypothesis, we inactivated retinogeniculate synapses originating from right eyes in Casp3^+/+ and Casp3^−/− mice with AAV-hSyn-TeTxLC injections (Fig. 6A). A group of animals was included for each genotype that received control injections. To measure synapse engulfment by microglia, we labeled RGC axon terminals with CTB conjugates and visualized microglia by immunostaining against Iba1, a marker specific for microglia in the brain (Fig. 6B-G). According to our model, in TeTxLC-injected Casp3^+/+ mice, microglia should preferentially engulf inactive retinogeniculate synapses originating from the right eye (Fig. 6A), whereas in TeTxLC-injected Casp3^−/− mice, synapses from the right and the left eye should be engulfed at similar levels regardless of their strength (Fig. 6A).

Figure 6.

To quantitatively detect engulfment of weak synapses by microglia, we compared TeTxLC-injected and control-injected animals of the same genotype (Fig. 6B, C, and F). We first calculated the ratio between the volume of right eye-originated RGC axon terminals and left eye-originated terminals that were engulfed by each microglia. Then, by comparing this right-to-left engulfment ratio in TeTxLC-injected vs. control-injected animals, engulfment of inactive synapses could be detected as higher engulfment ratios in TeTxLC-injected animals (Fig. 6F). It is worth noting confounding factors, such as surgical manipulations, cancel out when calculating engulfment ratios, allowing information pertaining only to substrate preference to be isolated.

Consistent with previous studies (7), in Casp3^+/+ mice inactivation of retinogeniculate synapses from the right eye led to enhanced microglia-mediated engulfment of right eye-originated RGC axon terminals but not left eye-originated terminals (Fig. S13A-B), resulting in a significant increase in right-to-left engulfment ratios in microglia of TeTxLC-injected animals compared to control-injected animals (Fig. 6B, C, and F). In contrast, engulfment ratios were similar between TeTxLC-injected and control-injected Casp3^−/− mice (Fig. 6D, E and G and Fig. S13C-D), suggesting that microglia could no longer distinguish between strong and weak synapses in the absence of caspase-3 activation. These observations remain valid when data were averaged and reported by animal (Fig. S13E-F). Summing the evidence obtained thus far, we propose the activity-dependent caspase-3 activation is a crucial signaling event that integrates upstream information about synaptic activity, takes place at weak/inactive synapses, and leads to activity-dependent elimination of weak synapses by microglia.

Caspase-3 deficiency protects against amyloid-β-induced synapse loss

Lastly, we wanted to test if caspase-3 regulates synapse loss in neurodegenerative diseases through mechanisms analogous to its role in activity-dependent synapse elimination. To this end, we focused on Alzheimer’s disease (AD). AD is the most common cause of dementia (50), and synapse loss is the strongest correlate of cognitive decline in AD patients (51). Accumulation and deposition of oligomeric or fibril amyloid-β (Aβ) peptide has been proposed to be the cause of neurodegeneration in AD (50). Intriguingly, oligomeric Aβ impairs long-term potentiation (LTP) (52), and this suppression of LTP requires caspase-3 activation (53). Additionally, caspase-3 activation has been linked to early synaptic dysfunction in an AD mouse model (54).

To test if synapse loss in AD is regulated by caspase-3, we utilized the APP/PS1 mouse line, in which amyloid precursor protein (APP) and presenilin 1 (PS1) carrying mutations associated with early-onset familial AD are overexpressed in CNS neurons (55). We observed that female App/Ps1^+/− mice developed amyloid deposits in the hippocampus and cortex by 5 months (Fig. S14Aii), and that the amyloid burden worsened by 6 months (Fig. S14Aiii). In male App/Ps1^+/− mice, amyloid deposits only became apparent at 6 months and occurred at a lower level compared to age-matched females (Fig. S14Aiv-v). We focused on analyzing App/Ps1^+/− female mice in subsequent experiments to obtain robust phenotypes. To quantify synapse loss, we labeled presynaptic and postsynaptic compartments in the dentate gyrus of 6 month-old App/Ps1^−/− and App/Ps1^+/− littermates by detecting synaptic vesicle protein 2 (SV2) and Homer1, respectively, and selected multiple fields of interest per animal for imaging analysis while avoiding regions adjacent to amyloid deposits (Fig. S15). We fitted ellipsoids to SV2 and Homer1 signals to identify pre- and post-synaptic puncta and defined synapses as Homer1-SV2 puncta pairs that were less than 300 nm apart (Fig. 7A). We observed that synapse density in App/Ps1^+/− mice was significantly reduced compared to that in App/Ps1^−/− littermate controls (Fig. 7A-B), and that this reduction could be attributed to lower densities of both pre- and post-synaptic puncta in App/Ps1^+/− mice (Fig. S16A-B). We then crossed APP/PS1 mice to caspase-3 deficient mice and quantified synapse density in 6 month-old Casp3^−/−; App/Ps1^−/− and Casp3^−/−; App/Ps1^+/− littermate mice (Fig. S15). We observed that, in the caspase-3 deficient background, overexpression of mutant APP and PS1 no longer resulted in a reduction in the density of synapses (Fig. 7C-D) or pre-/post-synaptic puncta (Fig. S16C-D), suggesting that caspase-3 activity is required for Aβ-induced synapse loss in the APP/PS1 mouse model. Intriguingly, levels of amyloid deposition were comparable between App/Ps1^+/− and Casp3^−/−; App/Ps1^+/− mice (Fig. S14Avi and B), and reactive microglia were observed near amyloid deposits regardless of caspase-3 deficiency (Fig. S17), indicating that protection conferred by caspase-3 deficiency against Aβ-induced synapse loss might not require inhibition of amyloid accumulation or microgliosis. We also investigated whether Aβ accumulation induced elevated levels of caspase-3 activity. We analyzed 4, 5, and 6 month-old App/Ps1^−/− and App/Ps1^+/− mice but only observed robust upregulation of caspase-3 activity in the dentate gyrus of a subset of 4 month-old App/Ps1^+/− mice (Fig. S18A-C). Upregulated caspase-3 activity in these mice remained in a punctate pattern without inducing neuronal death (Fig. S18D-F). It is possible that Aβ transiently induces caspase-3 activation in App/Ps1^+/− mice, but stochasticity in the onset and duration of such induction prevents reliable detection with our sample size and temporal resolution. Overall, our results demonstrate caspase-3 as an important regulator of Aβ-induced synapse loss and a potential therapeutic target for AD treatment.

Figure 7.

Discussion

A model for caspase-3 dependent synapse elimination

Our findings support a model where competitive interaction between strong synapses and weak synapses in the retinogeniculate pathway triggers caspase-3 activation in the postsynaptic compartments of weak synapses. Caspase-3 activation results in the removal of weaker synaptic connections by microglia, thereby facilitating the establishment of mature circuits in the visual pathway.

Mechanisms of activity-dependent caspase-3 activation

In the experiment where we demonstrated activity-dependent caspase-3 activation, we inactivated all retinogeniculate synapses from one eye to induce sufficiently high caspase-3 activity that can be discerned from background. Widespread caspase-3 activity induced by such strong inactivation at multiple sites in a given cell likely self-amplifies through positive feedback (35) and overwhelms negative regulation mechanisms (e.g., ubiquitin-dependent proteasomal degradation (24, 33, 34)), leading to the observed apoptosis of entire dLGN relay neurons (Fig. 1B). However, apoptotic neurons were infrequently observed in the dLGN of control animals (Fig. 1D and 2B), and caspase-3 deficiency does not significantly alter dLGN relay neuron numbers (Fig. S7), suggesting that during normal development, activity-dependent caspase-3 activation is predominantly localized and generally does not lead to neuronal death.

Intriguingly, synaptic inactivation in our experiment was achieved through TeTxLC expression in presynaptic RGCs, but caspase-3 activation was observed in postsynaptic relay neurons. Conversely, while caspase-3 is activated in the postsynaptic compartment, we were able to detect defects in synapse elimination using assays that measure presynaptic inputs. These observations suggest that activity-dependent caspase-3 activation requires synaptic transmission, and that caspase-3 dependent synapse elimination is not strictly axonal or dendritic but involves both pre- and post-synaptic compartments. An important limitation of this study is that experiments in full-body Casp3^−/− mice cannot query the roles of caspase-3 in presynaptic and postsynaptic compartments separately. Future experiments that perturb caspase-3 activation and other signaling events specifically in the pre- or post-synaptic compartments are needed to reveal more detailed mechanisms of activity-dependent synapse elimination.

The observation that activity-dependent caspase-3 activation requires the presence of both weak and strong synapses resonates with previous research demonstrating that relative synaptic efficacy biases the outcome of synapse elimination during development of neuromuscular junctions and the callosal pathway (15, 36, 56, 57). However, the mechanisms linking synaptic competition with caspase-3 activation remains elusive. During LTD, caspase-3 is activated through mitochondrial release of cytochrome-c (18). Whether synaptic competition could induce caspase-3 activation via mechanisms such as calcium signaling, which regulates both neurotransmission and cytochrome-c release (58), is an outstanding question.

Link between caspase-3 activation and synapse engulfment by microglia

How does caspase-3 activation at weak synapses lead to synapse engulfment by microglia? Caspase-3 activation is known to trigger the display of apoptosis markers that can be recognized by phagocytes (19). One of the best understood markers is phosphatidylserine (PtdSer), a phospholipid that translocates from the cytoplasmic leaflet of the plasma membrane to the exoplasmic leaflet during apoptosis (19). Importantly, caspase-3 directly regulates the enzymes that translocate PtdSer through enzymatic cleavage (59, 60). Two recent studies demonstrated that PtdSer is displayed on retinogeniculate synapses during postnatal development, and microglia use the exposed PtdSer as a cue to target and engulf synapses (8, 9). Whether PtdSer exposure is upregulated in response to synapse weakening and whether caspase-3 activity is required for such exposure are important questions that require further investigation.

The complement protein C1q has been observed to bind to PtdSer on apoptotic cells and facilitate phagocytic clearance (19). Additionally, a previous study found caspase-3 activity in C1q-positive synaptosomes (61), suggesting that C1q may bridge caspase-3 activation and synapse elimination. However, we investigated C1q expression and localization in the dLGN of P5 TeTxLC-injected mice but did not observe upregulation of C1q abundance or colocalization between C1q and caspase-3 activity (data not shown). It is often assumed in the literature that microglia directly engulf intact synapses, but existing data also support an alternative model where microglia indirectly contribute to synapse elimination by engulfing synaptic debris that is shed through neuron-autonomous mechanisms (62). The observation that synaptic activity promotes microglial engulfment through caspase-3 activation raises the possibility that weakened synapses partially disintegrate through apoptosis-like mechanisms prior to being engulfed by microglia. Further studies are needed to elucidate the intermediate steps between caspase-3 activation and microglial engulfment of synapses.

Caspase-3 as a potential target for AD treatment

Our findings suggest that caspase-3 deficiency confers protection against Aβ-induced synapse loss in a mouse model of AD. Notably, the absence of caspase-3 does not seem to affect Aβ deposition or microglia reactivity, indicating that caspase-3 depletion may mitigate synapse loss by directly countering Aβ-induced synaptic toxicity. Given the prominence of Aβ and inflammation as targets for AD drug development (63), inhibiting caspase-3 activity offers a potential alternative therapeutic strategy that could complement and enhance current treatment approaches. Therefore, it is important to test whether pharmacological inhibition of caspase-3 can effectively slow synapse loss and cognitive decline in both mouse models of AD and human patients.

Acknowledgements

We thank Crystall Lopez, Benjamin Gantz, and Brooke Groff for help with animal husbandry and breeding. We thank Sarah Lindo, Colin Morrow, Claire Boyer, and Rae Demars for help with surgery. We thank Monique Copeland, Amy Hu, Morgan Clarke, and Benjamin Foster for help with histology. We are also grateful to Sarah Kivimaki and the Viral Tools team of Janelia Core Services for help with AAV preparation. We thank Dr. Cagla Eroglu, Dr. Richard Axel, and Dr. David Clapham for comments on the manuscript. This work is supported by the Howard Hughes Medical Institute.

Additional files

Supplementary figures

Methods