Neural stem cells improve memory in an inducible mouse model of neuronal loss

Abstract

Neuronal loss is a major pathological outcome of many common neurological disorders, including ischemia, traumatic brain injury, and Alzheimer disease. Stem cell-based approaches have received considerable attention as a potential means of treatment, although it remains to be determined whether stem cells can ameliorate memory dysfunction, a devastating component of these disorders. We generated a transgenic mouse model in which the tetracycline-off system is used to regulate expression of diphtheria toxin A chain. After induction, we find progressive neuronal loss primarily within the hippocampus, leading to specific impairments in memory. We find that neural stem cells transplanted into the brain after neuronal ablation survive, migrate, differentiate and, most significantly, improve memory. These results show that stem cells may have therapeutic value in diseases and conditions that result in memory loss.

Figure 1.

Schematic of the CaM/Tet-DT_A-inducible transgenic system. RT-PCR data show DT_A expression can be induced and also abrogated by removal or readdition of doxycycline to the system, respectively. A, All mice, including pregnant females, were maintained on doxycycline (dox)-supplemented water or food to suppress transgene expression. Removal of dox from the drinking water or diet of CaM/Tet-DT_A mice alleviates transcriptional repression, leading to activation and expression of the DT_A transgene exclusively in CaMKIIα-expressing cells. B, RT-PCR shows that DT_A expression is first detectable by 8 d of induction (withdrawal of doxycycline). C, In mice induced for 11 d, DT_A is not detectable by RT-PCR after 7 d of reintroduction of doxycycline to the water.

Figure 2.

Induction of DT_A expression results in neuronal loss from hippocampal regions. A–D, No neuronal loss is apparent in noninduced mice as demonstrated by H&E and NeuN staining. E–H, Twenty days of induction leads to significant loss from the CA1 subfield. I–L, Thirty days of induction leads to widespread neuronal loss from the hippocampus. M, Loss of NeuN staining from the CA1 subfield is significantly decreased (*) from noninduced controls (NI) by 20 d of induction (20di) and exhibits further decline with 25 (25di) and 30 d of induction (30di) (ANOVA, F_(4,21) = 32.77, p < 0.0001, post hoc Bonferroni). N, Significant loss of neurons (*) from both the CA1 region and dentate gyrus at 30 d of induction is confirmed by unbiased stereological methods [CA1, t test, p = 0.0052; dentate gyrus (DG), t test, p = 0.0009].

Figure 3.

Length of induction affects the regional progression of neuronal loss. A, B, Fifteen days of induction results in loss localized to the CA1 region of the hippocampus (A), without corresponding loss from cortical regions (B). C–F, Twenty days of induction results in substantial loss from the CA1 subfield (C) with sparse loss from cortical regions (D), whereas 30 d of induction results in widespread loss from the CA1 region (E) and cortex (F).

Figure 4.

Hippocampal-dependent memory impairments. In mice induced for 20 d (20di) in which the majority of loss occurs within the CA1 region of the hippocampus, hippocampal-dependent place memory is significantly impaired (*), but cortical-dependent object memory is not significantly different from noninduced controls (NI). However, in mice induced for 30 d (30di), both place and object memory are significantly impaired (*), which is consistent with the extent of neuronal loss in both hippocampal and cortical areas. Noninduced CaM/Tet-DT_A mice are not significantly different from nontransgenic control mice. (place: one-way ANOVA, p = 0.035; NI vs 20di, p = 0.0187; NI vs 30di, p < 0.01; NI vs NonTg, p = 0.9452; object: one-way ANOVA, p < 0.0008; NI vs 20di, p > 0.05; NI vs 30di, p < 0.01; NI vs NonTg, p > 0.05).

Figure 5.

NSC transplants survive for at least 4.5 months and migrate and differentiate into neurons and glia. A, Paradigm of NSC transplantation in induced CaM/Tet-DT_A mice. B, Hippocampi at 4.5 months post-transplant of induced mouse showing NSCs (green) and neurons stained with NeuN (red). C, GFP NSCs transplanted into the hippocampus show migration differences based on microenvironment, with significantly more GFP+ cells migrating into cortical and white matter areas in noninduced mice compared with induced mice. D, Stereological quantification of GFP NSCs show significantly more GFP NSCs in the cortex (Ctx) of noninduced NSC-injected mice (*, cortex, t test, p = 0.05). E, F, GFP NSCs colocalize with NeuN (D), a marker of mature neuronal nuclei, GFAP-immunoreactive astrocytes (E), and CNPase (F), a marker for mature oligodendrocytes.

Figure 6.

NSCs improve hippocampal-dependent memory and increase synaptic density and neuronal number. Place and object memory in CaM/Tet-DT_A mice induced for 25 d at one month (A; p = 0.1583; p = 0.1311) and three months (B) post-transplant. At 3 months, induced mice with NSC transplants (IND NSC) show significant improvement in the hippocampal-dependent spatial task (*p = 0.0429) compared with induced vehicle injected controls (IND VEH) (p = 0.2137). C, Synaptic density in induced-vehicle injected vs induced-NSC injected mice at the stratum radiatum of the CA1 region and the polymorph layer of the dentate gyrus. D, A significant increase in synaptic density (*) is seen at the stratum radiatum of the CA1 subfield of NSC-injected induced mice [CA1 t test, p = 0.0316; dentate gyrus (DG), t test, p = 0.151]. E, There is also an increase in NeuN+ cells at the CA1 layer of induced NSC-injected mice compared with their vehicle-injected counterparts. F, This difference is significant (*) at both the CA1 region and the dentate gyrus (CA1 t test, p = 0.0035; DG, t test, p = 0.0099).