Treatment Trends: Tapping Stem Cells, DNA, and RNA to Save Neurons

Stem cells and RNA interference offer the latest and, to some, the greatest hope for novel therapies to fight neurodegenerative diseases. The prospect of replacing lost or dysfunctional neurons with stem cells, or knocking down pathogenic protein accumulation with RNAi, or even fixing disease-causing mutations by gene replacement drives the development of new technologies specifically for neurological diseases. A batch of recent studies dealing with all these possibilities provides a broad look at the promise of new treatments, and a reminder that the road to the clinic will not be a short one.

Stem cells to the rescue
Stem cells, often looked to as replacement parts for lost or faulty neurons, could play a completely different role in treating neurodegenerative diseases, according to a new study in the July 26 Journal of Neuroscience. The work, from Richard Sidman and colleagues at Harvard Medical School and Evan Snyder at the Burnham Institute for Medical Research, La Jolla, California, shows that rather than replace ailing neurons, implanted stem cells provide support for endogenous, at-risk neurons and preserve their health and function by modulating the tissue environment.

A major obstacle to using stem cells for neurodegenerative diseases has been the problem of how new neurons would be incorporated into functional circuits. The new results suggest that stem cells could ultimately find their use in preserving existing neurons and their connections rather than the considerably more challenging task of rebuilding networks after neurons have been destroyed. The stem cell results were obtained in a mouse model of inherited Purkinje neuron degeneration, but could be applicable to many other diseases, if treatment can be initiated early.

To study the effects of early stem cell transplantation, the researchers chose a well-defined model of neuron degeneration, the nervous mouse. Previously, first author Jianxue Li showed that in these mice, Purkinje cell death in the fourth to fifth postnatal week is triggered by a 10-fold increase in expression of tissue plasminogen activator (tPA), an important mediator of cerebellar development (Li et al., 2006). In the new study, Li and colleagues injected a neuron stem cell (NSC) line into the cerebellum during the first postnatal week, and found that Purkinje neurons are maintained in normal numbers and with normal morphology and connections right into adulthood. Cell transplants resulted in functional recovery as well—mice that received the transplants showed better motor performance on the rotarod, and this correlated with the number of rescued Purkinje neurons.

Somewhat unexpectedly, the bolstered neuronal complement did not derive from transplanted stem cells. The neurons did not express the β-galactosidase marker of the NSCs, and many of the neurons in female mice had detectable Barr bodies (an inactivated X chromosome), indicating that they derived from the host and not the donor line, which was male. The injected NSCs did differentiate, gaining neuronal cell makers and losing stem cell markers, but none stained with the Purkinje cell marker calbindin.

Consistent with the idea that NSCs provided a supportive function, transplantation reduced high brain levels of tPA protein and mRNA to normal. This normalization rectified downstream alterations in channel function, synaptic structure, and neurotrophic factor levels, all of which contributed to normalizing cell survival and function.

Just how NSCs can depress abnormal gene expression is not clear, but cell-cell contact appears to be important. The researchers showed that NSCs could rescue Purkinje cells in culture, but that neuron survival required direct contact between the cells. This was consistent with in vivo results showing that neuron rescue required NSCs to move into the parenchyma: Surviving Purkinje neuron number was positively correlated with integrated NSC number, but not with the number of NSCs in the superficial meninges.

The present study strengthens the concept of stem cells serving an underappreciated therapeutic role as chaperone cells’, the authors write, which may represent a more tractable therapeutic strategy for this neurogenetic disorder than the more conventionally considered goal of cell replacement. In most neural systems, as in the cerebellum, the prospect of reconstructing the complexity of connections and feedback loops beyond the periods of normal neurogenesis seems quite staggering. Preserving extant circuitry clearly would be more approachable.

The strategy of using stem cells to restore the neuronal environment and rescue host cells may also apply to other neurodegenerative disorders. Of course, treatment would need to start early before cells are totally lost, but the approach could be useful for slowly progressing diseases like Alzheimer disease, once the early stages can be readily identified.

A hit and a miss for RNAi
For many neurodegenerative diseases where the accumulation of misfolded, toxic proteins causes cell death, using RNA interference and antisense approaches to knock down the expression of specific proteins is a promising approach. That is exactly the path taken by Don Cleveland and colleagues at the University of California at San Diego, who report that delivery of synthetic antisense oligonucleotides directly into the CSF reduces levels of disease-causing SOD1 mutant mRNA and protein in the brain and spinal cord in a rat model of ALS. While the treatment did not delay onset of motor neuron disease, it did slow progression. The studies are a prelude to clinical trials in humans, which the researchers hope to start within a year, according to a press release on the study. The work, which was published online July 27 in the Journal of Clinical Investigation, offers a therapeutic strategy to downregulate almost any CNS protein.

One barrier to successful use of antisense oligos in the CNS is the problem of delivery. The researchers, led by joint first authors Richard Smith and Timothy Miller, tried delivering the oligos by osmotic pump into the cerebral ventricles. This method is already used in humans, they note, to deliver pain medication. Studies suggest that from the cerebral ventricles, the oligos would circulate in the cerebrospinal fluid, which bathes all regions of the CNS. Indeed, the authors showed that after a 14-day infusion in rats or rhesus monkeys, micromolar concentrations of oligonucleotides turned up in the brain parenchyma, as well as both the upper and lower regions of the spinal cord. They found oligonucleotides in both lumbar motor neurons and in non-neuronal cells.

Smith and colleagues then tested the ability of antisense SOD1 oligos to reduce protein levels in transgenic rats expressing the human G93A SOD1 mutant. These transgenic animals are a widely used ALS model. Even though the animals express very high levels of mutant SOD1 (5-10 times higher than endogenous SOD1), they effected a 40-60 percent reduction in mRNA and an approximately 25 percent reduction in protein when they administered a human-specific oligonucleotide. When they started infusion treatment in 65-day-old rats (~30 days before disease onset), they observed no change in onset, but the treatment did seem to slow progression, delaying the emergence of severe symptoms from day 122 to day 134, a 37 percent extension compared to the normal course of the disease.

Previous studies of viral delivery of antisense SOD1 in animals also showed promising results (Raoul et al., 2005; Ralph et al., 2005; Miller et al., 2005), but intraventricular delivery of oligonucleotides presents some advantages. First, the treatment can be easily regulated, with doses readily increased, reduced, or stopped. Also, in contrast to viral delivery schemes targeted only to neurons, oligonucleotides in the CNS get into other cells as well. This feature may be particularly important for ALS, where the expression of SOD1 in both neurons and surrounding glia is important in disease onset and progression (see ARF related news story and Boillee et al., 2006)

What of the application to other diseases When the researchers infused antisense oligonucleotides to two Alzheimer disease targets, presenilin-1 and GSK3β, they saw a reduction in mRNA for the two proteins in the right frontal/temporal cortex after 14 days. While this does not ensure that protein levels would be reduced, the results suggest that the widespread delivery of oligos throughout the CNS could open opportunities to treat a number of diseases.

Animal toxicity studies are now under way in preparation for a planned phase I of the SOD antisense oligo in humans. If the treatment proves safe, it could open up opportunities to knock down many more targets. The authors cite, for example, proteins such as the amyloid-β precursor protein, β-secretase, tau, and presenilin-1 for AD, or huntingtin for Huntington disease, as targets of interest for future studies.

Any future trials will have to consider the specter of side effects. For RNA interference therapies, the possibility of off-target effects raises safety concerns. In particular, a recent report showed that the administration of viral vectors that produce short hairpin RNAs (shRNAs, the precursors to small interfering RNAs) can be fatal to mice (Grimm et al., 2006): By overwhelming the cellular machinery that produces endogenous microRNAs, the shRNAs perturb gene expression generally, which leads to cell death.

Now, another paper, this one from Bernardo Sabatini and colleagues at Harvard Medical School, shows an additional off-target effect of shRNAs that is specific to neurons. In work published in the Journal of Neuroscience on July 26, Sabatini, Veronica Alvarez, and Dennis Ridenour show that expression of shRNAs in neurons interferes with dendritic spine structure and function, and can result in decreases in synapse number. (For a complete description of off-target effects of interfering RNAs, see the comment below from Zuoshang Xu.)

The effects are independent of which mRNA is targeted, and even of the generation of siRNAs, but do depend on the sequence of the shRNA. In particular, shRNAs that induced an antiviral-type response, as measured by activation of an interferon target gene, affected neuron morphology and function, while shRNAs to the same protein that did not induce the interferon gene, did not. For research, the results demonstrate the need to use caution in using RNA knockdown data to probe protein roles in synaptic function and remodeling. More careful sequence selection and use of stringent controls must be the norm—the use of scrambled sequences is not sufficient, the authors say, and they stress the need for protein rescue experiments to ultimately prove the specificity of any observed effects.

For therapeutic RNAs, the same cautions will apply. However, synthetic antisense DNA oligonucleotides may have an advantage over shRNAs because, as Cleveland and colleagues argue, they do not require further processing and do not trigger an antiviral response.

Corrective measures
Finally, what about gene therapy—a permanent fix for those genetic errors that give rise to many neurodegenerative diseases For Alzheimer, Parkinson, and Huntington diseases, and others whose familial forms spring from dominant gain-of-function mutations, the strategy of adding a good copy of the gene will not work. Instead, the goal of gene therapy must be to actually repair the mutations in the gene in situ. While novel methods have been devised for therapeutic recombination in cells (see ARF related news story), the ultimate goal is to find techniques that work in vivo.

A new approach that uses adeno-associated viral vectors comes closest yet to this elusive prize. In a report published online in Nature Biotechnology, researchers from David Russell’s lab at the University of Washington in Seattle used the vector to repair a model mutation in a β-gal reporter gene in mouse liver in vivo. The group, led by first author Daniel Miller, also repaired a disease-causing mutation in the GusB gene encoding the liver enzyme β-glucuronidase, although the frequency of repair (one to two cells per 10,000) was too low to demonstrate a therapeutic effect. For that enzyme, they estimate a 10-fold increase in the efficiency of recombination would achieve a clinical effect. Though gene replacement in the CNS will be many times harder than correcting liver enzymes, the work provides a tantalizing promise of what may be possible, one day.—Pat McCaffrey.


Li J, Imitola J, Snyder EY, Sidman RL. Neural stem cells rescue nervous Purkinje neurons by restoring molecular homeostasis of tissue plasminogen activator and downstream targets. J Neurosci. 26 July 2006;26:7839-7848. Abstract

Smith RA, Miller TM, Yamanaka K, Monia BP, Condon TP, Hung G, Lobsiger CS, Ward CM, McAlonis-Downes, M, Wei H, Wancewicz EV, Bennett CF, Cleveland DC. Antisense oligonucleotide therapy for neurodegenerative disease. Journal of Clinical Investigation. July 27 2006; online publication. Abstract

Alvarez VA, Ridenour DA, Sabatini BL. Retraction of synapses and dendritic spines induced by off-target effects of RNA interference. J. Neurosci. July 26 2006;26:7820-7825. Abstract

Miller DG, Wang P, Petek LM, Hirata RK, Sands MS, Russell DW. Gene targeting in vivo by adeno-associated virus vectors. Nature Medicine. July 30 2006; advance online publication. Abstract

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