Honolulu: Neuroregeneration Taps Glia, Neurotrophins

Series - Alzheimer's Association International Conference on Alzheimer's Disease 2010:

Over the years, hopes of using neural stem cells to patch wide swaths of failing brain circuitry in Alzheimer disease have met mostly with skepticism. However, new data from several independent studies suggest these approaches may have a fighting chance, and that their heroics rest less with making new neurons and more with boosting glial cells and neurotrophins. At the International Conference on Alzheimer’s Disease (ICAD) held 10-15 July 2010 in Honolulu, Hawaii, Maria Grazia Spillantini, University of Cambridge, U.K., reported that neural precursor cells, which become astrocytes and oligodendrocytes after transplantation, prevent cortical neurodegeneration in a mouse model of human tauopathy. These findings, just published in the July 28 Journal of Neuroscience, struck a chord with recent work by the lab of Frank LaFerla, University of California, Irvine, who spoke at ICAD on the latest tweaks to his approach using neural stem cells to replenish synapses and restore cognition in AD transgenic mice. Meanwhile, a strategy based on a neurotrophic peptide reverses memory loss and drives neurogenesis in the same AD strain (3xTg) and in a mouse model of Down syndrome, according to studies presented by Inge Grundke-Iqbal and colleagues at New York State Institute for Basic Research, Staten Island. The time seems ripe to focus on regenerative approaches, particularly with glial cells, as the first clinical trial using human embryonic stem cells gained approval by the U.S. Food and Drug Administration last week (see The New York Times story). The experimental therapy will use oligodendrocyte precursors to treat patients with spinal cord injury.

On the preclinical front, Spillantini and colleagues began exploring neuroprotective strategies after discovering age-related, cortical cell loss in a tauopathy mouse model they had created and characterized previously as having a largely motor phenotype (Allen et al., 2002). These transgenic mice express mutated (P301S) human tau and, at two to three months of age, start to develop a range of motor phenotypes (e.g. poor grip, crossed hind limbs, tremor, muscle weakness) that become severely disabling by five to six months. At this point, P301S mice have lost half their motor neurons and show extensive aggregation of hyperphosphorylated tau (Delobel et al., 2008). However, it was initially unclear whether the massive neuronal death extended into cortical regions, prime areas of destruction in human frontotemporal dementias.

Using cresyl violet and NeuN staining to count neurons in the cerebral cortex of P301S transgenic and age-matched wild-type mice, first authors David Hampton and Daniel Webber, of the University of Cambridge and now at the University of Edinburgh, found no appreciable cell loss in the tau transgenics at two months. However, three-month-old P301S mice had pronounced neuronal death, and “very few cells left” by five months, Spillantini told the ICAD audience. While this mimics human disease, “no one knows how the cells died,” she said. “In human studies, all you have is end-stage tissue to analyze.”

In the P301S mice, however, the researchers were able to stain cortical tissue with the phosphorylation-dependent anti-tau antibody AT8 at various time points to track pathological changes accompanying the cell loss. They saw ring-like tau staining at two months, increased cell body and dendritic signal at three months, and many tau inclusions by five months. Among neurons with ring-like tau deposits, some died before tau tangles appeared, while others didn’t seem to form tangles at all, Spillantini reported.

The tangle-free cells seemed salvageable, prompting the scientists to test whether a cell-based approach could curb the neuronal demise in the tauopathy model. After all, recent rodent studies suggest that astrocyte precursors can help in motor neuron disease (Lepore et al., 2008 and ARF related news story; Yamanaka et al., 2008). Moreover, LaFerla’s lab has used neural stem cells to improve memory in 3xTg AD mice (Blurton-Jones et al., 2009 and ARF related news story). This mouse line develops both amyloid and tau pathologies without obvious motor deficits, making it amenable to cognitive tests that require motor function.

Spillantini and colleagues tried a similar strategy in the P301S tauopathy mice, injecting fluorescent neural stem cells into their cortex at two months of age, and analyzing the transplant region one or three months later. The stem cell transplants brought neuron counts in the tau transgenics up to wild-type levels, as judged by NeuN staining at both time points. Glial cells seemed to mediate these effects, as the vast majority of transplanted cells did not become neurons but instead differentiated into astrocytes and oligodendrocytes. This jibes with LaFerla’s study, which found hardly any neurons among the progeny of the stem cells transplanted into 3xTg mice. Furthermore, when Spillantini and colleagues cultured neural stem cells and pushed them toward the astrocytic lineage in vitro, they found that transplantation of the pre-differentiated astrocytes rescued cell death in the tauopathy mice just as the stem cell transplants had done before.

Consistent with other studies demonstrating bystander effects of neuroprotection (Nagahara et al., 2009 and ARF related news story), cells derived from the transplanted neural precursors expressed high levels of glial cell-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) mRNA, as judged by quantitative PCR (qPCR). Brain-derived neurotrophic factor (BDNF) levels looked normal by qPCR, but were elevated, along with those of nerve growth factor, in immunohistochemical staining of brain tissue from stem cell-transplanted mice. Other studies, including LaFerla’s stem cell transplantations in 3xTg mice, have identified BDNF as a mediator of neuroprotection. Directly injecting the neurotrophin into the brains of transgenic mice helped them do better on spatial memory tests, whereas BDNF-depleted stem cells failed to improve the animals’ cognition (Blurton-Jones et al., 2009 and ARF related news story).

At ICAD, LaFerla and first author Mathew Blurton-Jones presented, in separate talks, the latest developments in this ongoing work. The researchers had shown that the neural stem cells could rescue cognition one month after transplantation, but wonder whether the neuroprotection would hold three to six months later and beyond, as the mice continued racking up brain Aβ. In other words, “if you don’t get rid of that amyloid, is that a bad thing? Does it matter? We’d been thinking it’s a bad thing,” LaFerla told ARF. Studies to determine if the cognitive benefits wane after the first month post-transplantation are ongoing.

In the meantime, acting on a hunch that the benefits may fade with time, the scientists have stably expressed neprilysin, an Aβ-degrading enzyme, in neural stem cells. This gives the cells a one-two punch, as they not only make a neurotrophic factor, but also churn out a protein that helps slow amyloid pathology within transplanted animals. In preliminary studies, the neprilysin-expressing stem cells markedly reduced plaque load in aged 3xTg mice. Knowing the approach reduces Aβ, the team has begun a new set of longitudinal behavioral studies to ask whether the neprilysin provides additional benefit or longer-lasting effects on cognition in the AD mice, Blurton-Jones told ARF. Importantly, the effects so far seem to extend beyond the transplanted region, opening the door in the future for introducing neuroprotective factors via the periphery rather than brain injections. “If the neprilysin works, you may be able to modify mesenchymal cells or blood cells to express neprilysin and hopefully have that circulate through the brain,” LaFerla speculated.

Julie Blanchard, working with Grundke-Iqbal, described a pharmacological approach for neuroregeneration in mice that does involve peripheral injection, though not of engineered cells but of a peptide that spurs neuronal differentiation of endogenous progenitor cells. Grundke-Iqbal’s lab has synthesized a brain-permeable 11-mer (aka peptide 6) corresponding to the active region of CNTF and shown previously that it enhances memory in wild-type mice (Chohan et al., 2009). At ICAD, Blanchard reported that daily intraperitoneal injection of this peptide for six weeks restored not only short- and long-term memory, but also neurogenesis, and dendritic and synaptic plasticity, in seven- to eight-month-old 3xTg AD mice. Like LaFerla’s stem cell studies in the same mouse strain, the CNTF peptide had no effect on Aβ or tau pathology, Blanchard said. Her 3xTg findings were accepted last week for publication in the journal Acta Neuropathologica.

On a poster, Grundke-Iqbal reported that 30-day slow release of the peptide in the form of subcutaneous pellets was able to restore neurogenesis and cognition in 11- to 15-month-old Ts65Dn Down syndrome mice. Adult neurogenesis also goes awry in the Ts1Cje mouse model of Down syndrome, which has fewer neural progenitors and an excess of astrocytes, according to a report published July 28 in PLoS One (Hewitt et al., 2010). Taken together, these recent studies “further support the notion that trophic-based therapies should be aggressively pursued,” Blurton-Jones said.—Esther Landhuis

Comments

  1. The effects of BDNF on neuronal protection and synapse preservation are most impressive, similar to those shown by NGF. As they do not cross the blood-brain barrier (BBB), new methods are tested, such as the stem cell injection or genetic vector transfer of genes that induce increased release of BDNF or NGF. Clearly, that limits the application of these growth factors.

    If I may draw attention to the fact that there are other growth factors out there that actually cross the BBB and show exactly the same protective effects: I presented a poster at the meeting on the effects of the incretin GLP-1 analogue Liraglutide. Incretins are well known as novel treatments for type 2 diabetes, but really are growth factors that have numerous other properties in addition to regulating insulin signaling. Liraglutide not only crosses the BBB, but also protects memory in APP/PS1 mice, protects LTP, and reduces plaques and the associated inflammation response in the brain (as assessed by measurement of activated microglia) when injected peripherally. Interestingly enough, the drug is already on the market as a treatment for type 2 diabetes (Victoza, by NovoNordisk).

    This suggests that there is no need to transfer stem cells or gene vectors into the brain by intracerebroventricular injection if a growth factor analogue can be given peripherally.

    References:
    Abstract at the ICAD: Liraglutide, a novel GLP-1 analogue, prevents the impairment of learning and LTP in an APP/PS-1 mouse model of Alzheimer's disease. McClean PL, Parthasarathy V, Gault VA, Hölscher C.

    View all comments by Christian Hoelscher

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References

News Citations

  1. Baby Steps Toward Cell Therapies for ALS
  2. Support Cast: Neural Stem Cell BDNF Prompts Memory in AD Mice
  3. BDNF the Next AD Gene Therapy?

Paper Citations

  1. Allen B, Ingram E, Takao M, Smith MJ, Jakes R, Virdee K, Yoshida H, Holzer M, Craxton M, Emson PC, Atzori C, Migheli A, Crowther RA, Ghetti B, Spillantini MG, Goedert M. Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. J Neurosci. 2002 Nov 1;22(21):9340-51. PubMed.
  2. Delobel P, Lavenir I, Fraser G, Ingram E, Holzer M, Ghetti B, Spillantini MG, Crowther RA, Goedert M. Analysis of tau phosphorylation and truncation in a mouse model of human tauopathy. Am J Pathol. 2008 Jan;172(1):123-31. PubMed.
  3. Lepore AC, Rauck B, Dejea C, Pardo AC, Rao MS, Rothstein JD, Maragakis NJ. Focal transplantation-based astrocyte replacement is neuroprotective in a model of motor neuron disease. Nat Neurosci. 2008 Nov;11(11):1294-301. PubMed.
  4. Yamanaka K, Chun SJ, Boillee S, Fujimori-Tonou N, Yamashita H, Gutmann DH, Takahashi R, Misawa H, Cleveland DW. Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis. Nat Neurosci. 2008 Mar;11(3):251-3. PubMed.
  5. Blurton-Jones M, Kitazawa M, Martinez-Coria H, Castello NA, Müller FJ, Loring JF, Yamasaki TR, Poon WW, Green KN, Laferla FM. Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease. Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13594-9. PubMed.
  6. Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, Wang L, Blesch A, Kim A, Conner JM, Rockenstein E, Chao MV, Koo EH, Geschwind D, Masliah E, Chiba AA, Tuszynski MH. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer's disease. Nat Med. 2009 Mar;15(3):331-7. PubMed.
  7. Chohan MO, Li B, Blanchard J, Tung YC, Heaney AT, Rabe A, Iqbal K, Grundke-Iqbal I. Enhancement of dentate gyrus neurogenesis, dendritic and synaptic plasticity and memory by a neurotrophic peptide. Neurobiol Aging. 2011 Aug;32(8):1420-34. PubMed.
  8. Hewitt CA, Ling KH, Merson TD, Simpson KM, Ritchie ME, King SL, Pritchard MA, Smyth GK, Thomas T, Scott HS, Voss AK. Gene network disruptions and neurogenesis defects in the adult Ts1Cje mouse model of Down syndrome. PLoS One. 2010;5(7):e11561. PubMed.

Other Citations

  1. 3xTg

External Citations

  1. The New York Times story

Further Reading

Papers

  1. Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, Wang L, Blesch A, Kim A, Conner JM, Rockenstein E, Chao MV, Koo EH, Geschwind D, Masliah E, Chiba AA, Tuszynski MH. Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer's disease. Nat Med. 2009 Mar;15(3):331-7. PubMed.
  2. Blurton-Jones M, Kitazawa M, Martinez-Coria H, Castello NA, Müller FJ, Loring JF, Yamasaki TR, Poon WW, Green KN, Laferla FM. Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease. Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13594-9. PubMed.
  3. Chohan MO, Li B, Blanchard J, Tung YC, Heaney AT, Rabe A, Iqbal K, Grundke-Iqbal I. Enhancement of dentate gyrus neurogenesis, dendritic and synaptic plasticity and memory by a neurotrophic peptide. Neurobiol Aging. 2011 Aug;32(8):1420-34. PubMed.

Primary Papers

  1. Hampton DW, Webber DJ, Bilican B, Goedert M, Spillantini MG, Chandran S. Cell-mediated neuroprotection in a mouse model of human tauopathy. J Neurosci. 2010 Jul 28;30(30):9973-83. PubMed.

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