21 Oct 2022

In the October 19 Science Advances, researchers led by Michel Cayouette, McGill University, Montreal, reported that the trafficking protein Numb tempers tau tangles in mouse retinal ganglion cells and in motor neurons. In tauopathy mice, overexpressing Numb slowed neurodegeneration, while knocking out Numb increased soluble tau, spurred axon blebbing, and killed neurons, accelerating hind limb paralysis. “This study implicates Numb, an important member of the Notch signaling pathway, as a novel regulator of intracellular pathological tau,” wrote Kiran Bhaskar, University of New Mexico, Albuquerque (comment below).

Notch signaling controls cell differentiation and intracellular protein trafficking. As one member of this cascade, Numb regulates neuronal migration and growth in the central nervous system, but its role in the adult CNS is less well studied. Alternative splicing carves Numb into four isoforms—weighing in at 65, 66, 71, or 72 kDa. Two have a short phosphotyrosine-binding domain, and two a long domain. Removal of a proline-rich region shortens these by another six kDa (see image below). Previously, scientists found a link between Numb and amyloid precursor protein (APP). They found that neurons expressing isoforms with the short PTB, namely Numb-65 or Numb-71, accumulated more APP in early endosomes than neurons expressing Numb-66 or Numb-72 (Kyriazis et al., 2008). First author Marine Lacomme and colleagues wondered if Numb also regulates tau.

Numb Splicing. The trafficking protein Numb comes in four flavors, each with a short or long version of the phosphotyrosine-binding domain (PTB) and the proline-rich region (PRR). [Courtesy of Lacomme et al., Science Advances, 2022.]

To find out, Lacomme expressed each Numb isoform along with human tau in human kidney cells. All four isoforms co-immunoprecipitated with tau, indicating they interact, but only Numb-72 lowered soluble tau concentration. The researchers then transfected this isoform into cultured RGCs from P301S tau mice and from 3xTg animals, which carry P301L tau plus APP and presenilin mutations. RGCs accumulate tau and degenerate in tauopathies, and the eye offers easy access to the CNS. While axons of tauopathy RGCs typically bleb up and the cells struggle to regulate intracellular calcium, overexpressing Numb-72 prevented both. To the authors, these results suggested that Numb-72 tempered tau, normalized neuron signaling, and staved off neurodegeneration. Indeed, while a mild excitotoxic stress in the form of the NMDA injection into the eye halved the number of RGCs in 6-month-old P301S and in 6-month-old 3xTg mice, intraocular injections of a Numb-72-toting virus that targets RGCs protected the ganglia.

Numbing Neurons. Compared to healthy neurons (top left), neurons lacking Numb (top right) accumulate tau, their axons bleb up, and they die. Tauopathy neurons (bottom left) undergo the same process. However, overexpression of Numb-72 (bottom right) reduces tau aggregation, normalizes axons, and increases cell survival. [Courtesy of Lacomme et al., Science Advances, 2022.]

Overexpression can introduce artifacts, but the scientists corroborated these observations by conditionally knocking out Numb in RGCs of wild-type mice. In these knockouts, RGCs accumulated fivefold more total tau than did controls. Tau oligomers jumped 10-fold, and twice as many blebs appeared on the axons. By 20 months, the Numb knockouts had half as many RGCs as controls. Ditto for 8-month-old offspring of Numb knockouts crossed with P301S mice. Notably, these P301S/Numb KO mice had 20 percent fewer RGCs than either P301S mice or wild-type Numb knockouts, indicating a synergism between the P301S mutation and Numb in controlling intraneuronal tau (see image below).

Numb’s effects spanned beyond RGCs. Because the gene driver used in the knockouts also turns off Numb in spinal motor neurons, the scientists could study these neurons, too. Motor neurons degenerate in P301S mice, leading to hind limb paralysis by 7 to 10 months old. By 11 months, P301S/Numb KOs had two-thirds fewer spinal motor neurons than P301S mice and their hind limbs had deteriorated. P301S mice were paralyzed by 1.5 years old, but in Numb knockouts this occurred six months earlier.

How does Numb temper tau? It ramps up extracellular tau release, claim the authors. HEK cells overexpressing Numb-72 cranked out eight times as much total tau into the medium than did wild-type cells, while the amount of extracellular oligomeric tau did not change. Cultured RGCs from P301S mice overexpressing Numb-72 released 2.5-fold more tau than their unaltered P301S counterparts.

The authors believe that, by speeding the secretion of tau monomers, Numb-72 minimizes the amount available for aggregation, which spares the cells. “This interesting hypothesis seems to disagree with other hypotheses that stipulate that tau secretion might itself be ultimately neurotoxic,” wrote Scott Small, Columbia University (comment below). Bhaskar agreed and was curious if the secreted tau monomers acted as seeds for tau propagation (comment below).—Chelsea Weidman Burke

Comments

1. • Kiran Bhaskar
     University of New Mexico
   • Posted: 21 Oct 2022

   This is truly a tour de force in terms of different methodologies and models they have used to conclude that Numb (especially 72 kDa isoform) interacts with tau, regulates intracellular tau levels, and provides neuroprotection in different model systems.

   The main takeaway from this study is that for the first time it implicates an important member of the Notch signaling pathway (which are typically involved in cell fate decision and cellular trafficking) as a novel regulator of intracellular pathological tau. It clearly demonstrates how enhancing Numb levels could bring down toxic tau levels inside the cell and it shows this is neuroprotective. They also nicely demonstrate that knocking down Numb does the opposite in retinal ganglion cells. This conclusion is based on solid sets of data both in vitro and in vivo model systems.

   There are lot of interesting aspects to this study, but to me, it was interesting to note that Numb (72) maintains intracellular tau homeostasis by secreting soluble (mostly monomeric) tau into the extracellular space to keep intracellular tau level well-balanced. This observation opens up more questions, including whether this extracellularly secreted tau acts as a “seed” for tau propagation (although the authors don’t seem to see any changes in the level of tau phosphorylation). This aspect needs further exploration. Nevertheless, this study identifies Numb as a novel regulatory to pathogenic tau and could serve as potential therapeutic target.

   View all comments by Kiran Bhaskar
2. • Scott Small
     Columbia University
   • Posted: 21 Oct 2022

   What distinguishes AD-associated tauopathy from other tauopathies is accelerated active tau secretion. This process does not occur via the conventional secretory pathway but is suggested to occur via “unconventional” endolysosomal secretion; namely upon the fusion of late-endosomal/multivesicular bodies with the cell surface.

   Besides supporting this mode of tau secretion in AD, this elegant study suggests how tau secretion might be linked to intraneuronal tau pathology and its neurotoxicity. Specifically, the authors interpret their findings to suggest that the endolysosomal pathway primarily secretes tau monomers, resulting in fewer intraneuronal tau oligomers, which are proposed to be more neurotoxic. 

   This interesting hypothesis seems to disagree with others that stipulate tau secretion itself might ultimately be neurotoxic. Future studies might disambiguate these hypotheses by blocking endolysosomal secretion and testing whether this worsens or ameliorates tau-related toxicity.

   View all comments by Scott Small

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References

Research Models Citations

1. Tau P301S (Line PS19)
2. 3xTg

Paper Citations

1. Kyriazis GA, Wei Z, Vandermey M, Jo DG, Xin O, Mattson MP, Chan SL. Numb endocytic adapter proteins regulate the transport and processing of the amyloid precursor protein in an isoform-dependent manner: implications for Alzheimer disease pathogenesis. J Biol Chem. 2008 Sep 12;283(37):25492-502. PubMed.

Further Reading

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