Machuca-Parra AI, Bigger-Allen AA, Sanchez AV, Boutabla A, Cardona-Vélez J, Amarnani D, Saint-Geniez M, Siebel CW, Kim LA, D'Amore PA, Arboleda-Velasquez JF. Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL. J Exp Med. 2017 Aug 7;214(8):2271-2282. Epub 2017 Jul 11 PubMed.
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University of Edinburgh
University of Southern California
Machuca-Parra and colleagues present an interesting study showing that Notch3 signaling is linked to vascular smooth muscle cells (VSMCs) coverage in retinal vessels and demonstrate that restoring Notch3 signaling via genetic rescue and using a Notch3 agonist antibody (A13) prevents small vessel disease (SVD) phenotype (e.g., VSMC loss) in both mouse models of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and in Notch3 knockout (KO) mice.
Pathology of brain vascular mural cells, including both VSMCs and pericytes, is central to both CADASIL patients and transgenic CADASIL models. The authors examined the α-smooth muscle actin (SMA)-positive VSMCs coverage in the retina and found reduced VSMCs coverage in Notch3 KO mice that can be rescued genetically by re-expressing wild-type Notch3. In contrast, re-expression of CADASIL C455R mutant in Notch3 KO mice neither rescued VSMCs nor contributed to VSMCs loss. These observations raise a couple of questions. What is the impact of Notch3 on the pericyte cell population covering brain capillaries? Are the effects restricted to the SMA-positive VSMCs pool? A previous report found no changes associated with VSMCs, but reported a significant decrease in pericytes attributed to Notch aggregation in a mouse CADASIL model carrying the R169C Notch3 mutation (Ghosh et al., 2015). Although it is known that Notch3 may regulate the expression of standard pericyte markers (i.e., PDGFRβ, NG2, and Desmin), investigation of the pericyte population lacking SMA in the brain by using a conditional Notch3 KO mouse model be the next step toward new insights into the role of mural cells and toward understanding the significance of Notch3 signaling in CADASIL.
Furthermore, the authors provided evidence that the A13 antibody is efficient in HEK cells in vitro and can cross the blood-retinal barrier to reach the perivascular space. However, the only in vivo evidence demonstrating that mural cells can be activated by A13 administration is the presence of active cleaved Notch3 in brain vessels, which suggests no antibody uptake by mural cells but a direct binding. Further analyses have to be completed in regard to the distribution of Notch3 in vessels to understand how restoration of Notch3 signaling contributes to VSMC versus pericyte function in CADASIL mice.
Finally, although the authors studied the retina for obvious reasons, further studies need to be performed on the brain parenchyma, especially in white matter (WM) regions such as the corpus callosum and the internal capsule because of the association between CADASIL and WM disease. Is it possible to restore Notch3 signaling and prevent mural cell loss in those specific WM structures using A13 antibody? And importantly, is the WM integrity preserved in A13-treated as compared to control IgG-treated CADASIL mice?
References:
Ghosh M, Balbi M, Hellal F, Dichgans M, Lindauer U, Plesnila N. Pericytes are involved in the pathogenesis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. Ann Neurol. 2015 Dec;78(6):887-900. Epub 2015 Oct 7 PubMed.
View all comments by Berislav ZlokovicQB3 University of California Berkeley
This well-designed study takes two big steps forward for the CADASIL field by (1) showing a therapeutic effect of a Notch 3-activating antibody in a mouse model, and (2) providing evidence for the potential utility of the Notch 3 extracellular domain as a translatable disease biomarker.
I think the CADASIL field has argued long enough about whether the disease results from gain versus loss of Notch 3 function. When arguments like this go on for decades, the answer is usually “both.” It’s nice to see this paper move past that tangle and begin translating known biology to therapy.
The potential clinical significance of these findings is even greater given recent evidence that Notch 3 mutations may be a major contributor to cerebral small vessel disease in the general population (see, for example, Rutten et al., 2016). In addition, given that small vessel disease often coexists with and likely exacerbates Alzheimer’s pathology (Kisler et al., 2017) and vice versa, it would be of interest to look for beneficial effects of the Notch 3-activating antibody in Alzheimer’s models.
References:
Rutten JW, Dauwerse HG, Gravesteijn G, van Belzen MJ, van der Grond J, Polke JM, Bernal-Quiros M, Lesnik Oberstein SA. Archetypal NOTCH3 mutations frequent in public exome: implications for CADASIL. Ann Clin Transl Neurol. 2016 Nov;3(11):844-853. Epub 2016 Sep 28 PubMed.
Kisler K, Nelson AR, Montagne A, Zlokovic BV. Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease. Nat Rev Neurosci. 2017 Jul;18(7):419-434. Epub 2017 May 18 PubMed.
View all comments by Gabrielle LeblancSchepens Eye Research Institute and Harvard Medical School
I appreciate the comment by Drs. Zlokovic, Zhao, and Montagne on our work addressing the role of Notch3 signaling in mural cell loss.
They raised important questions:
I agree this is a relevant point that was not addressed in the current publication, and concur that the issue will be properly resolved using a conditional Notch3 KO mouse model allowing for abrogation of Notch3 signaling in specific mural cell subpopulations. We have recently generated such a model and have begun preliminary analyses.
Unambiguous analyses of the brain vasculatures will require characterization of complete vasculatures similar to what we have accomplished in the retina. This may be done by implementing tissue-clearing methodologies such as CLARITY.
References:
Chung K, Deisseroth K. CLARITY for mapping the nervous system. Nat Methods. 2013 Jun;10(6):508-13. PubMed.
View all comments by Joseph Arboleda-VelasquezImago Pharmaceuticals
This study is an important advance for CADASIL. CADASIL patients have no available therapies. They desperately need increased awareness of this terrible disease and an interest from researchers in developing disease-modifying drugs.
Dr. Arboleda-Velazquez's works suggests that antibodies that activate the genetic target of CADASIL, Notch3, could play an important therapeutic benefit in CADASIL models. Hopefully these studies continue to advance and translate into benefit for CADASIL patients.
View all comments by Irene Griswold-PrennerMcMaster University
This is a very exciting study. These results add an interesting perspective on the still-controversial question of the mutational spectrum of CADASIL (see for examples Rutten et al., 2013, and Moccia et al., 2015). Perhaps study of additional mutations, using the same experimental design, will help further address this question. This would also help delineate the therapeutic potential of the A13 antibody for treatment and prevention of CADASIL.
Well done!
References:
Rutten JW, Boon EM, Liem MK, Dauwerse JG, Pont MJ, Vollebregt E, Maat-Kievit AJ, Ginjaar HB, Lakeman P, van Duinen SG, Terwindt GM, Lesnik Oberstein SA. Hypomorphic NOTCH3 alleles do not cause CADASIL in humans. Hum Mutat. 2013 Nov;34(11):1486-9. Epub 2013 Oct 7 PubMed.
Moccia M, Mosca L, Erro R, Cervasio M, Allocca R, Vitale C, Leonardi A, Caranci F, Del Basso-De Caro ML, Barone P, Penco S. Hypomorphic NOTCH3 mutation in an Italian family with CADASIL features. Neurobiol Aging. 2015 Jan;36(1):547.e5-11. Epub 2014 Aug 27 PubMed.
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