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.

Serum from young mice enhanced synapses and activity in human neurons.

SPARCL1 and THBS4 were more abundant in serum from young than old mice.

These proteins mediated the synaptic effects of young serum.

“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 “This supports unpublished findings by us at Alkahest and others that young plasma or specific fractions and proteins can overcome aging associated deficits,” she wrote.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