Abstract
Arshad MN, Oppenheimer S, Jeong J, Buyukdemirtas B, Naegele JR. Neurobio Dis. 2022;174:105879. doi:10.1016/j.nbd.2022.105879 GABAergic interneurons play a role in regulating adult neurogenesis within the dentate gyrus (DG) of the hippocampus. Neurogenesis occurs within a stem cell niche in the subgranular zone (SGZ) of the DG. In this niche, populations of neural progenitors give rise to granule cells that migrate radially into the granule cell layer (GCL) of the DG. Altered neurogenesis in temporal lobe epilepsy (TLE) is linked to a transient increase in the proliferation of new neurons and the abnormal inversion of Type 1 progenitors, resulting in ectopic migration of Type 3 progenitors into the hilus of the DG. These ectopic cells mature into granule cells in the hilus that become hyperexcitable and contribute to the development of spontaneous recurrent seizures. To test whether grafts of GABAergic cells in the DG restore synaptic inhibition, prior work focused on transplanting GABAergic progenitors into the hilus of the DG. This cell-based therapeutic approach was shown to alter the disease phenotype by ameliorating spontaneous seizures in mice with pilocarpine-induced TLE. Prior optogenetic and immunohistochemical studies demonstrated that the transplanted GABAergic interneurons increased levels of synaptic inhibition by establishing inhibitory synaptic contacts with adult-born granule cells, consistent with the observed suppression of seizures. Whether GABAergic progenitor transplantation into the DG ameliorates underlying abnormalities in adult neurogenesis caused by TLE is not known. As a first step to address this question, we compared the effects of GABAergic progenitor transplantation on Type 1, Type 2, and Type 3 progenitors in the stem cell niche using cell type-specific molecular markers in naïve, non-epileptic mice. The progenitor transplantation increased GABAergic interneurons in the DG and led to a significant reduction in Type 2 progenitors and a concomitant increase in Type 3 progenitors. Next, we compared the effects of GABAergic interneuron transplantation in epileptic mice. Transplantation of GABAergic progenitors resulted in reductions in inverted Type 1, Type 2, and hilar ectopic Type 3 cells, concomitant with an increase in the radial migration of Type 3 progenitors into the GCL. Thus, in mice with Pilocarpine induced TLE, hilar transplants of GABA interneurons may reverse abnormal patterns of adult neurogenesis, an outcome that may ameliorate seizures.
Commentary
Temporal lobe epilepsy (TLE) is among the most common forms of epilepsy. About 30% of these patients do not attain seizure control with medications. 1 This leads to reduced independence, persistent fear of having a seizure, higher doses of combinations of medications that cause unwanted side effects, and increased mortality in the form of sudden unexpected death in epilepsy, or SUDEP. 2 Novel and improved treatment options are needed to control seizures in these patients. Even better, would be development of ways to prevent or mitigate the changes that lead to epileptogenesis. Temporal lobe epilepsy is thought to arise from some inciting event (eg, febrile seizure, in utero insult, injury, etc) that initiates a cascade of cellular, molecular, and anatomical changes that ultimately result in excitation-inhibition imbalance and development of spontaneous, unpredictable seizures. 3 Animal models exist that recapitulate this series of events well. These include the post-status epilepticus (SE) models of TLE caused by pilocarpine (Pilo-TLE) or kainic acid. 4 The excitotoxicity from the prolonged seizure sets off similar changes in the rodent hippocampus and leads to epilepsy. Among these changes, abnormal proliferation of neuroprogenitor cells and aberrant adult neurogenesis in the dentate gyrus promote epileptogenic circuit reorganization, such as ectopic granule cells, mossy fiber sprouting, and hilar basal dendrites. There are 3 types of neuroprogenitor cells. Type 1 are radial glia-like progenitors that send processes that pass through the granule cell layer (GCL) to terminate in the molecular layer. These give rise to Type 2 progenitors, which then give rise to Type 3 progenitors, or neuroblasts, that differentiate into dentate granule cells. Type 1 progenitor cells become inverted such that they no longer send radial projections from the sub granular zone (SGZ; the site of neurogenesis) to the GCL but instead send projections to the hilus. 5 This leads to ectopic migration of Type 3 progenitors to the hilus, and epileptogenic hyperexcitability.
An intriguing potential way to prevent or reverse development of seizures after an insult is to transplant stem cells into key brain regions. Stem cell transplants are being studied for therapeutic use for several conditions. 6 Prior work in mice shows that transplant of GABAergic progenitors in the dentate gyrus of the hippocampus reduces seizures in the Pilo-TLE model. 7 How exactly this happens is not well-understood. GABAergic progenitor transplantation has been shown to directly increase GABAergic inhibition in the dentate gyrus, but another possibility is by triggering circuit reorganization in the hippocampus to restore normal progenitor migration and neurogenesis. In the current study, Arshad and colleagues examined this question. 8
Fetal GABAergic progenitor cells were obtained from the median ganglionic eminence of E13.5 embryos and implanted into hippocampus of mice that had undergone Pilo-TLE or had undergone sham treatment. They then examined anatomical changes in hippocampus in TLE and control mice that received transplant or that received cell-free media as control.
First, they found that GABAergic progenitor transplantation into the hilus reduced the number of inverted Type 1 neuronal progenitors in TLE mice, compared to TLE mice that received cell-free media. Since inverted Type 1 progenitors lead to ectopic migration of granule cells, this suggests that ectopic migration should be reduced. There was no effect on overall number of Type 1 progenitors. Type 1, Type 2, and Type 3 progenitors were differentiated by expression of molecular markers and location within the hippocampus. Inverted Type 1 progenitors were identified by projection pattern.
Second, they found that transplantation of GABAergic progenitors led to an increase in adult-born dentate granule cells, regardless of whether the mouse had undergone TLE or not. But it appears to have occurred to a greater extent in TLE mice. Interestingly, the total number of Type 3 progenitors in the GCL was increased following GABAergic progenitor transplantation. Upon further evaluation, there was a significant reduction in ectopic Type 3 progenitors in the hilus in TLE mice with progenitor transplantation compared to those with media injection. This suggests that ectopic migration, which would otherwise contribute to hyperexcitability, is inhibited, and normal migration to the GCL is supported by GABAergic progenitor transplantation in the hilus.
Third, GABAergic progenitor transplantation also led to an increase in adult-born neurons in the dorsal hippocampus, but not in the ventral hippocampus. This suggests that the effects of transplant are limited to the regions juxtaposed to the transplant site, which could have important practical implications. Transplantation of GABAergic progenitors did not affect cell proliferation in the SGZ of the dentate gyrus in either TLE or control mice. This was assessed by counting bromodeoxyuridine (BrdU)-positive cells 2 hours after injection in mice 1 week following transplantation. Because the TLE mice also received midazolam to abort SE, whereas control mice did not, they performed an experiment to determine whether midazolam alters cell proliferation and found that it did not. However, GABAergic progenitors increased the proportion of adult-born neurons that survived in both TLE and control mice, compared to media injected animals. Again, survival, as assessed by BrdU expression, was increased in the dorsal hippocampal regions near the transplant site compared to the more distant ventral regions.
This is a well-executed and important study demonstrating restoration of normal Type 1 progenitor orientation and appropriate migration of Type 3 progenitors in the GCL instead of to the hilus, which could account, at least in part, for the beneficial effects of GABAergic progenitor transplantation in normalizing GABAergic inhibition in the hippocampus and reducing seizure-promoting hyperexcitability. Now that they have established that these changes occur, more work will be needed to directly test the contribution of these various changes to inhibition of seizures and understand how these changes occur. Much of this seems to depend on where the Type 1 progenitors project, which will dictate where adult-born cells migrate (ie, GCL vs hilus). Proliferation of Type 1 progenitors is dependent on the subset of GABAergic neurons expressing parvalbumin. This study did not differentiate between types of GABAergic neurons driven by the progenitor transplants. In this study there were some unavoidable confounds. One they addressed by testing for effects of midazolam in seizure-naïve animals. One that was not empirically addressed, but was addressed with literature review, is that the TLE animals were singly housed whereas control mice were group housed, raising the possibility of a role for social isolation in the effects seen in TLE animals.
Looking a little further down the road, who will be the ideal candidates for these types of therapies must be considered. Patients that have TLE, but that are not candidates for surgical resection due to the location of the lesion in eloquent tissue could be one possibility. Another candidate group could be patients at elevated risk for developing TLE based on a known precipitating injury. Could similar therapies using stem cells to normalize seizure-facilitating circuity be useful in other types of epilepsy, such as those caused by a discrete focal lesion? As the specific mechanisms for the changes brought about by transplantation are better elucidated, perhaps there will be ways to manipulate the underlying mechanisms and exact the same changes without the use of stem cells which can be controversial. 9,10 Of course, one way this could be done would be through gene-editing, which can be similarly controversial, though editing autologous induced pluripotent stem cells could be a possibility. 10 While this clearly elucidates parts of the mechanism to reduce excitability in rodents, there is some controversy regarding whether and to what extent adult neurogenesis occurs in human patients with TLE. 11,12 There may be some dependence on age, or on underlying etiology for TLE. Translatability will depend on determining how closely adult neurogenesis in humans resembles that in rodents. From an ethical standpoint, it is important to consider where tissue for transplant would come from for use in patients. 10 Continued work will be needed on this front to develop appropriate reagents. Regardless, this work in mice is promising and continues us down the path to potential novel therapies for epilepsy.
