Ever since mammalian adult neurogenesis was confirmed, scientists had hoped to get pluripotent cells of the subventricular zone (SVZ) to become something other than olfactory bulb neurons when they mature. That hope was recently realized when researchers managed to persuade neural progenitors to migrate and become striatal, cortical, and substantia nigra neurons, among others. In fact, in some cases new neurons have been found to send axons to appropriate targets and may even contribute to functional recovery (see Nakatomi et al., 2002 and Scharff et al., 2000).
But what about other types of neurons, for example, the motor neurons that are lost in devastating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), otherwise known as Lou Gehrig’s disease? In a study published this week in the early online edition of PNAS, Jeffrey Macklis's team at Massachusetts General Hospital and Harvard University demonstrate that SVZ neural precursors can, indeed, be induced to make the long trek up to the motor cortex of the adult mouse brain where they replace corticospinal motor neurons. Even more exciting is the fact that these new neurons manage to send axonal projections all the way to the spinal cord.
The appearance of new adult neurons in areas outside the olfactory bulb and the dentate gyrus of the hippocampus has been shown to occur in response to various insults, including stroke and epilepsy. In fact, researchers have been able to induce neurogenesis with apoptosis and ischemia. Such damage apparently upregulates developmental signals that induce pluripotent cells in the subventricular zone to begin their trek away from the ventricles, and that guide these soon-to-be neurons to the appropriate cortical layers (see, for example, Wang et al., 1998).
Macklis's team was one of the first to successfully use this approach to coax SVZ stem cells to replace neurons in the anterior neocortex; the new cells even made appropriate axonal connections with the thalamus (see ARF related news story). In the current study, Macklis, first author Jinhui Chen, and Sanjay Magavi employ a variation of their previous methodology to replace neurons even farther from the proliferative zone.
The researchers initiated the death of corticospinal motor neurons by injecting "Trojan horse" compounds into the spinal cord. After several weeks, when these fluorescent nanospheres—and their nefarious cargo of chlorin e6, a porphyrin derivative which releases singlet oxygen when illuminated—had been transported back along the corticospinal axons to the cell bodies of layer V in the motor cortex, the authors induced apoptosis by photoactivating the chlorin. Then, to track new neurons, they added BrdUrd, which labels dividing cells, into the drinking water and let the mice sip the compound for two weeks.
Microscopic analysis showed that the Trojan horses selectively targeted the corticospinal motor neurons (CSMNs), and that the irradiation caused degeneration of 10-20 percent of these cells. When Chen and colleagues examined the brains of the mice two weeks after induction of apoptosis, they found that new neurons, staining positive for BrdU and the neuronal marker doublecortin, appeared in layer V of the cortex in treated animals but not in controls. In fact, the authors were able to detect 20-30 new neurons per cubic millimeter in treated mice, but no new neurons in mice that were not irradiated.
Doublecortin is a marker of early neuronal development. When Chen and colleagues used laser scanning confocal microscopy to track doublecortin neurons, they found that the protein was expressed only within about two weeks of the original apoptotic insult. After that, cells began expressing the later neuronal marker NeuN, and they took on a more mature morphology. Again, confirming the selectivity of the process, Chen found NeuN- and BrdU-positive neurons only in regions that had undergone ablation of CSMNs. In fact, maturation of the neurons was evident by the gradual increase in NeuN expression; the BrdU:NeuN ratio rose from about 25, two weeks after apoptosis, to about 35 two weeks later, but then fell to less than 10 when the animals were 12 weeks old. More than one year after induction of apoptosis, the authors found that adult-born neurons were still present.
That these neurons took up residence only in the appropriate cortical layer, exhibited adult pyramidal neuron morphology, and did not express markers inappropriate to corticospinal motor neurons is strong evidence that this process may be physiologically normal. But are any of these new neurons functional?
To answer this, the authors used the retrograde tracer FG. When injected into the spinal cord, this label travels back up the motor neuron axons to the cell bodies. Chen found that FG labeled both old and new CSMNs, indicating that the newcomers are making projections to the spinal cord. One fascinating aspect of this experiment is that it demonstrates how slowly new connections can be made. At eight weeks after apoptosis, no new connections could be observed, but at 12 and 16 weeks, the authors did find new neurons labeled by the tracer. Over one year later (56 weeks post-apoptosis), Chen could record 1-7 new neurons per cubic mm making contact projections with the spinal cord neurons.
“Together, these results demonstrate that targeted apoptosis of CSMNs leads to microenvironmental change that recruits immature neurons to the cortex in a spatially and temporally specific manner,” state the authors.
One reason that these results are so promising is that, unlike other approaches such as stem cell transplants into the brain, this targeted killing of cells does not perturb the microenvironment of the cortical cells. There is no inflammation, gliosis, or microglial activation. The success of this study and its predecessors also suggests that preserving the microenvironment may be key to promoting the unimpeded growth of axons to their targets.—Hakon Heimer
- Nakatomi H, Kuriu T, Okabe S, Yamamoto S, Hatano O, Kawahara N, Tamura A, Kirino T, Nakafuku M. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell. 2002 Aug 23;110(4):429-41. PubMed.
- Scharff C, Kirn JR, Grossman M, Macklis JD, Nottebohm F. Targeted neuronal death affects neuronal replacement and vocal behavior in adult songbirds. Neuron. 2000 Feb;25(2):481-92. PubMed.
- Wang Y, Sheen VL, Macklis JD. Cortical interneurons upregulate neurotrophins in vivo in response to targeted apoptotic degeneration of neighboring pyramidal neurons. Exp Neurol. 1998 Dec;154(2):389-402. PubMed.
- Chen J, Magavi SS, Macklis JD. Neurogenesis of corticospinal motor neurons extending spinal projections in adult mice. Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16357-62. PubMed.