A new recipe for cooking up cholinergic neurons from stem cells may help researchers study what goes wrong with these cells in Alzheimer’s disease. In the April 21 Nature Biotechnology online, researchers led by Su-Chun Zhang at the University of Wisconsin, Madison, report that their method generates forebrain-specific cholinergic neurons. This cell type plays a crucial role in learning and memory. Moreover, when transplanted into mice with cholinergic lesions, the neurons reversed memory deficits. This suggests that the cells are functional and will be useful in research, Zhang said. A more tantalizing and still unanswered question is whether transplanting such neurons could eventually hold promise as a therapy for AD or other cognitive disorders.
“It’s quite an exciting paper. It ties together previous studies connecting the cholinergic system with memory to the more recent capacity to derive cholinergic neurons from stem cells, and shows that you can recover memory function by improving cholinergic function in the hippocampus,” said Ole Isacson of Harvard Medical School’s McLean Hospital, Belmont, Massachusetts. He was not involved in the work.
Cholinergic activity in the basal forebrain falters early in Alzheimer’s disease, and this dysfunction correlates with cognitive deficits (see, e.g., ARF related news story). Three of the four approved AD medications enhance cholinergic signaling by inhibiting the breakdown of the neurotransmitter acetylcholine. In addition, previous studies showed that fetal tissue grafts containing cholinergic neurons improved memory in aged and lesioned rats (see Gage et al., 1984; Leanza et al., 1998; and Cassel et al., 2002). More recently, researchers have devised recipes to make cholinergic neurons from stem cells (see ARF related news story; Bissonnette et al., 2011). However, these methods had limitations. In particular, these cholinergic neurons expressed only some of the markers seen in basal forebrain, suggesting they might not represent the subtype of cholinergic cell involved in memory.
To develop specific forebrain cells, first author Yan Liu differentiated human embryonic stem cells into neuroepithelial cells, and then exposed them for two weeks to 1,000 ng/ml of sonic hedgehog, a developmental protein factor that instructs cells to assume a ventral forebrain identity. The resulting cells resembled ventral forebrain progenitors called medial ganglionic eminence (MGE) cells. The authors then added nerve growth factor (NGF), a survival factor for cholinergic neurons. Four days later, about 90 percent of the cells had become neurons. Of these, almost 40 percent contained the cholinergic enzyme ChAT, while 45 percent appeared to be GABAergic interneurons. After a few weeks in vitro, cholinergic cells were electrically active and expressed several markers of mature basal forebrain cholinergic neurons.
Next, the authors moved to mice. First, they created a memory deficit by injecting a toxin into the septum between the left and right hemispheres. This wiped out all of the cholinergic cells in the vicinity. Then they transplanted human MGE-like progenitors into the hippocampus. Six months later, most of the grafted cells were mature neurons. Half were GABAergic, while 8 percent, or about 12,000 cells per graft, expressed cholinergic markers. Those cholinergic neurons appeared to integrate into the local circuitry, extending fibers along the hippocampal molecular layer. Their axons expressed synaptic proteins and appeared to make contact with dendrites of hippocampal pyramidal neurons, suggesting they were forming synapses. Recordings from brain slices showed that the cholinergic neurons were electrically active.
The acid test for a neuron, however, is whether it can support brain function. The new cells passed this test, too. Animals with transplants performed better in the Morris water maze and passive avoidance tests than did lesioned controls. The improvement began at two months after transplant, and was statistically significant at four and six months—the longest timepoint tested.
As an additional control, some lesioned mice received transplants of ventral spinal progenitors (VSPs). These cells produce similar numbers of cholinergic and GABAergic neurons in culture as do MGE cells, but the neurons are committed to a spinal identity and do not express forebrain markers. VSP grafts produced few cholinergic cells, did not integrate into the hippocampus, and VSP-treated animals performed poorly in the functional tests.
These divergent results show that not just any kind of cholinergic or GABAergic neuron can contribute to learning and memory, Zhang told Alzforum. “It’s the unique type of cholinergic and GABAergic neurons that belong to the basal forebrain,” he said. In ongoing work, Zhang is generating forebrain cholinergic neurons from induced pluripotent stem cells made from people with AD and Down’s syndrome. He believes these cells could help researchers study what goes wrong in patients. The neurons might also be useful for in-vitro drug screening, Zhang suggested. In addition, he is collaborating with other groups to transplant his MGE-like progenitors into several AD mouse models to see what effects they have in a disease environment.
Could grafts of these precursor cells be a treatment for people with AD or other disorders that affect the cholinergic system? Elliott Mufson at Rush University, Chicago, Illinois, sounded a note of caution, pointing out that many questions remain as to whether the transplanted cells made functional, physiological connections in this paradigm. Future work could look at synapses by electron microscopy and investigate whether the new neurons innervated appropriate target cells, Mufson suggested. He recalled that grafts sometimes behave in unexpected ways; for example, some Parkinson’s patients who received fetal dopaminergic transplants experienced debilitating side effects (see ARF related news story). “I’m hesitant to say this is a potential treatment for neurodegenerative disease,” Mufson said.
Isacson found the treatment implications exciting. He pointed out that cholinergic activity fades in normal aging as well as in AD (see Strong et al., 1980), and its loss also correlates with cognitive deficits in Parkinson’s disease (see Perry et al., 1985). If researchers had a means to identify specific subpopulations of AD or PD patients who had a cholinergic deficit, then transplants could potentially help them, Isacson suggested. Several groups are working on positron emission tomography (PET) tracers specific for cholinergic neurons that might enable researchers to detect problems in this system (see, e.g., Kovac et al., 2010; Giboureau et al., 2010).—Madolyn Bowman Rogers
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