11 Jun 2019

Human neurons sprouted more synapses and fired off more action potentials when bathed in serum from young, but not old, mice, according to a study published June 3 in Proceedings of the National Academy of Sciences. Kathlyn Gan and Thomas Südhof, Stanford University, zeroed in on two proteins—SPARC-like protein 1 (SPARCL1) and thrombospondin-4 (THBS4) that bestowed serum with these synaptogenic powers. The researchers did not test whether these two proteins enhance synapses in vivo, or whether they cross the blood-brain-barrier. Even so, the findings raise the question if circulating proteins could influence neuronal activity and function.

“Overall the study validates that protein factors in serum derived from young, but not aged mice can affect critical neuronal functions that are impaired in aging,” commented Eva Czirr of Alkahest, San Carlos, California. The company is developing plasma products to counter brain aging. “This supports unpublished findings by us at Alkahest and others that young plasma or specific fractions and proteins can overcome aging associated deficits,” she wrote. She would not comment if SPARCL1 or THBS4 are components of GRF6019, a plasma fraction Alkahest is testing in a Phase 2 trial for AD. 

Ever since parabiosis experiments showed that blood from young animals could slow brain aging in old ones, researchers have tried to identify the specific factors involved (Nov 2009 news; May 2014 news). In mice, TIMP-2, a metalloproteinase inhibitor in human umbilical cord blood, and growth differentiation factor 11 (GDF11) from the blood of young mice both increased neuronal plasticity (Apr 2017 news; Ozek et al., 2018). On the other hand, scientists pegged eotaxin 1 as a brain aging accelerator, and VCAM1 on endothelial cells as an accomplice that allows aging factors to tax the brain (Dec 2018 news; May 2019 news). All these studies were done in whole mice.

Gan tested serum directly on neurons. She differentiated human neurons from embryonic stem cells, and co-cultured them with mouse glia for support. She then supplemented the growth medium with fetal bovine serum (FBS), serum from 15-day-old mice, or serum from 12- to 15-month-old mice. About a month later, she examined the synapses.

Synapses Sprout. Human neurons exposed to serum from young mice (right) had more synapses (green, synapsin) than neurons grown in serum from old mice (middle) or fetal bovine serum (left). [Courtesy of Gan et al., PNAS, 2019.]

Neurons grown in the presence of young mouse serum had 30 percent more dendritic branches, 65 percent more synapses, and twice as many dendritic spines. They spontaneously generated twice as many miniature excitatory postsynaptic potentials (mEPSPs) as did neurons grown in FBS or old mouse serum. They evoked higher amplitude synaptic responses through AMPA and NMDA receptors, and recruited more NMDA receptors to the synapse.

Notably, old mouse serum did not crimp the number of function of synapses compared with FBS, suggesting it contained no inhibitory factors. To test this further, Gan cultured neurons in either young or old mouse serum for 25 days, then switched out half of their medium with the “opposite” medium, containing either young or old serum, for another week. She found that young serum strongly boosted synaptic function even in neurons previously grown in old serum, while adding old serum to neurons previously grown in young serum only modestly decreased synaptic function. Together, these findings suggested that young serum contained factors that boosted synaptic function, even in the presence of old serum.

What gave young blood its oomph? Using mass spectrometry, Gan identified six proteins that were more than 1.5-fold enriched in young versus old serum. They investigated two—SPARCL1 and THBS4—which were two and four times higher, respectively, in young serum. Subsequent experiments revealed that SPARCL1 and THBS4 recapitulated many of the benefits of young serum, including increases in dendritic arborization, synaptic density, and spontaneous synaptic responses. SPARCL1, but not THBS4, also tripled NMDA receptor-mediated activity. These benefits persisted even when the scientists added the proteins to neurons cultured in medium supplemented with serum from old mice.

Overall, the findings suggest that SPARCL1 and THBS4 enhance synaptic function in human neurons, but whether they contribute to the rejuvenating effects of young blood in old brains remains to be seen. Both proteins have been cast as astrocyte factors involved in synaptogenesis, but they are also widely expressed throughout the body, where they help shape the extracellular matrix (Christopherson et al., 2005; Kucukdereli et al., 2011; Mosher and Adams, 2012). Why these proteins decrease with age in mouse serum and how they enhance human neuronal synapses needs to be investigated.

Do the proteins decline with age in human serum? Do they cross the blood-brain barrier to make contact with neurons in the brain? Saul Villeda of the University of California, San Francisco, commended the study’s use of human neurons. He said that while it is unclear if it will translate in vivo, is the study is an important step in identifying potential synapse-promoting factors. Future experiments could inject tagged versions of SPARCL1 and THBS4 into mice to see if they enter the brain and influence synaptic function, Villeda said. Human organoid systems could also lend insight into how these proteins might function in the human brain (Jun 2019 news). Villeda also noted that the study does not address how systemic factors sway other cells in the brain as they age, including microglia.—Jessica Shugart

Comments

1. • Eva Czirr
     Confluence Therapeutics
   • Posted: 11 Jun 2019

   It is exciting to read a paper focusing on potential mechanistic aspects of young plasma-mediated regeneration of the aging brain. In these elegantly designed studies, the focus lies on how synapse generation and synaptic function in human ES-cell-derived neurons are impacted by serum from young and aged mice. The authors clearly demonstrate a direct beneficial effect of young mouse plasma-derived serum on neurons, even in the absence of glia cells.

   Interestingly, not only was young serum beneficial on its own, but it was able to overcome effects of aged serum when it was added after cells were 35 days in vitro and miniature excitatory post-synaptic currents were recorded at DIV42. Conversely, aged serum had the opposite effect, suggesting that young serum might also make the neurons more resistant to detrimental effects. Using TMT Mass Spec the authors identified THBS4 and SPARCL1 as specific factors enriched in young serum and lacking in aged serum as mediators of beneficial effects on synapse formation and function.

   Overall, the study validates that protein factors in serum derived from young, but not aged mice can affect critical neuronal functions that are impaired in aging. This supports findings by us at Alkahest (unpublished data) and others (Villeda et al., 2011; Villeda et al., 2014; Castellano et al., 2017) that young plasma or specific fractions and proteins can overcome aging associated deficits.

   References:

   Villeda SA, Luo J, Mosher KI, Zou B, Britschgi M, Bieri G, Stan TM, Fainberg N, Ding Z, Eggel A, Lucin KM, Czirr E, Park JS, Couillard-Després S, Aigner L, Li G, Peskind ER, Kaye JA, Quinn JF, Galasko DR, Xie XS, Rando TA, Wyss-Coray T. The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature. 2011 Sep 1;477(7362):90-4. PubMed.

   Villeda SA, Plambeck KE, Middeldorp J, Castellano JM, Mosher KI, Luo J, Smith LK, Bieri G, Lin K, Berdnik D, Wabl R, Udeochu J, Wheatley EG, Zou B, Simmons DA, Xie XS, Longo FM, Wyss-Coray T. Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice. Nat Med. 2014 Jun;20(6):659-63. Epub 2014 May 4 PubMed.

   Castellano JM, Mosher KI, Abbey RJ, McBride AA, James ML, Berdnik D, Shen JC, Zou B, Xie XS, Tingle M, Hinkson IV, Angst MS, Wyss-Coray T. Human umbilical cord plasma proteins revitalize hippocampal function in aged mice. Nature. 2017 Apr 19; PubMed.

   View all comments by Eva Czirr

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References

News Citations

1. Chicago: The Vampire Principle—Young Blood Rejuvenates Aging Brain? 20 Nov 2009
2. In Revival of Parabiosis, Young Blood Rejuvenates Aging Microglia, Cognition 5 May 2014
3. Umbilical Cord Blood Tied to Hippocampal Rejuvenation 20 Apr 2017
4. How Immune Cells From Blood Beget Aging in Brain 14 Dec 2018
5. Paper Alert: VCAM1 Opens the Door to Brain Aging 18 May 2019
6. Reproducible Brain Organoids Could Offer New Models for Research 6 Jun 2019

Paper Citations

1. Ozek C, Krolewski RC, Buchanan SM, Rubin LL. Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice. Sci Rep. 2018 Nov 23;8(1):17293. PubMed.
2. Christopherson KS, Ullian EM, Stokes CC, Mullowney CE, Hell JW, Agah A, Lawler J, Mosher DF, Bornstein P, Barres BA. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell. 2005 Feb 11;120(3):421-33. PubMed.
3. Kucukdereli H, Allen NJ, Lee AT, Feng A, Ozlu MI, Conatser LM, Chakraborty C, Workman G, Weaver M, Sage EH, Barres BA, Eroglu C. Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC. Proc Natl Acad Sci U S A. 2011 Aug 9;108(32):E440-9. Epub 2011 Jul 25 PubMed.
4. Mosher DF, Adams JC. Adhesion-modulating/matricellular ECM protein families: a structural, functional and evolutionary appraisal. Matrix Biol. 2012 Apr;31(3):155-61. Epub 2012 Jan 14 PubMed.

External Citations

1. Phase 2 trial

Further Reading

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