Too Much of a Good Thing—A Toxic Aβ Blocker?

The endoplasmic reticulum protein reticulon-3 (RTN3) can prevent amyloid-β plaques—but only if it doesn’t become aggregated itself, according to a paper in the July 22 Journal of Neuroscience. RTN3 holds BACE1, which participates in the production of Aβ, in the endoplasmic reticulum (ER) where the neutral pH prevents it from cleaving amyloid precursor protein (APP). The study authors, based at the Cleveland Clinic Foundation in Ohio, found that RTN3 was protective in a mouse model of Alzheimer disease, but can itself form aggregates, distorting neurons and negating its positive effects. Principal investigator Riqiang Yan hopes that if he can find a way to block RTN3 aggregation, augmenting RTN3 activity would have potential as a therapy for AD.

Reticulons are ER residents involved in membrane morphology and intracellular trafficking. Reticulons have also been linked to neurologic diseases including epilepsy (Bandtlow et al., 2004) and amyotrophic lateral sclerosis (Fergani et al., 2005). RTN4, also called Nogo, inhibits neurite outgrowth after injury and is mislocalized in the AD brain (Park et al., 2006). Increased levels of any reticulon expression reduces Aβ levels (He et al., 2004; Murayama et al., 2006). Yan and colleagues have found that RTN3 is a major constituent of dystrophic neurites in the AD brain (Hu et al., 2007), but this finding is yet to be confirmed by other researchers. Wataru Araki’s group at the National Institute of Neuroscience in Tokyo found little difference in RTN3 expression between AD and control brain samples, although they did see it co-localize with BACE1 (Kume et al., 2009). The discrepancy, Yan suggested, could be due to different RTN3 antibodies, which can produce different staining patterns.

Yan and colleagues have engineered a mouse model that overexpresses human RTN3 along with the endogenous mouse gene. In these animals, the protein appears to accumulate in and swell dendrites and axons in what the researchers call RIDNs—RTN3 immunoreactive dystrophic neurites. Earlier this year the Yan group reported that RIDNs also form in the brains of aged, nontransgenic mice (Shi et al., 2009). In the current work, first author Qu Shi, Yan and colleagues crossed their RTN3 mouse with a common AD model expressing mutant APP and presenilin (Tg-PA mice; Borchelt et al., 1997) to create a triple transgenic line they called Tg-R3PA.

Tg-PA mice normally have amyloid plaques by six months of age. Compared to the double mutants, the Tg-R3PA animals had fewer, and smaller, plaques in the cerebral cortex, presumably because RTN3 blocked BACE1’s cleavage of APP to produce Aβ. In Tg-PA mice, 0.3 percent of the cortex was occupied by Aβ; only 0.1 percent was plaque-covered in Tg-R3PA mice. Cortical levels of Aβ1-40 and Aβ1-42 were also reduced in Tg-R3PA animals, according to sandwich ELISAs. Yet in the hippocampus, the effect of RTN3 was diminished, with no significant difference in plaque load between the two lines.

Though the cortex and hippocampus produce similar amounts of RTN-3, the authors had previously shown that the hippocampus is more prone to RIDN formation. Specifically, the CA1 region contains the highest levels of neurites damaged by RTN-3. When they analyzed subregions of the hippocampus, Shi and colleagues found that the Tg-R3PA mice had fewer plaques than Tg-PA animals in both the CA3 region and dentate gyrus; only the CA1 area was unprotected in the triple transgenics. Since aggregated RTN-3 does not interact with BACE1 (He et al., 2006), the scientists suspect that in the CA1 region, aggregated RTN-3 forms dystrophic neurites and is unable to block APP cleavage. “We still don’t know why CA1 is the most susceptible region,” Yan said.

The researchers also used cell culture models to probe the interactions between RTN-3 and BACE1, comparing normal HEK-293 cells to a line stably expressing RTN3, called HR3M. In subcellular fractionation experiments, they found that more BACE1 was in the endoplasmic reticulum in HR3M cells than in control cultures. BACE1 requires an acidic pH to cleave APP—an environment it might find in an endosome or secretory vesicle, but not in the ER. Indeed, full-length APP levels were higher in the HR3M line.

Without BACE1, Aβ production is limited, and reducing the protein’s levels diminishes pathology in mouse AD models (see ARF related news story on Singer et al., 2005; McConlogue et al., 2007). Increasing RTN3 levels or activity might do the same, the authors suggest—if only one could also block aggregation and RIDN formation. “We believe if we can inhibit the aggregation, we may reduce the formation of those dystrophic neurons,” Yan said. In addition, fewer aggregates might mean more monomers to block BACE1.

“I suspect that excess RTN3 expression in their transgenic mice is responsible for the abnormal RTN3 aggregate formation,” Araki wrote in an e-mail to ARF. Perhaps that much RTN3 is unnecessary to prevent amyloid production: “If lower expression of RTN3 suffices to inhibit Aβ accumulation, induction of RTN3 may have therapeutic value.”—Amber Dance

Comments

    • User Profile Image Riqiang Yan
      University of Connecticut Health
    • Posted:

    First of all, I greatly appreciate Amber Dance for writing this news story. I would like to take this opportunity to add a few clarifications to this study. Finding abundant reticulon 3 (RTN3) aggregates accumulated in dystrophic neurites is indeed quite interesting. We have demonstrated that our RTN3 antibody (R458) marks abundant dystrophic neurites in surrounding amyloid plaques, and we named this population of dystrophic neurites as RTN3 immunoreactive dystrophic neurites (RIDNs). Angela Chang in Bruce Trapp’s lab (Cleveland Clinic) used the same antibody and replicated the same observation using postmortem brain samples from a different source (personal communication). We have noticed that detection of these abundant RIDNs may require an antibody that will recognize an epitope of aggregated RTN3 (discussed further below). Our antibody R459, which recognizes only the RTN3 monomer, detects only small numbers of dystrophic neurites in surrounding plaques. Stephen Strittmatter’s RTN4 (Nogo) antibody can also mark dystrophic neurites in human AD postmortem brain (Park et al., 2006).

    More interestingly, we found that transgenic mice overexpressing myc-tagged RTN3 driven by a murine prion promoter, a system developed by David Borchelt, develop abundant and dispersed RIDNs. Ultrastructural examination of RIDNs performed by Allan Levey’s group at Emory University revealed the presence of a large amount of protofibril-like aggregates near the axonal terminus as shown in our EMBO J article (Hu et al., 2007). Immuno-electron microscopy (EM) experiments, also performed at Emory University, suggest that the aggregates are enriched within membrane-enclosed structures whose sizes range from 3-5 μm, consistent with swollen neurites (Hu et al., 2007). A pre-embedding immuno-EM experiment performed by Xinghua Yin in the Trapp lab showed compacted and densely immunoreactive signals accumulated within a swelling axon (Hu et al., 2007).

    We initially had a concern as to whether RIDNs formation in RTN3 transgenic mice was due to an overexpression artifact or because of a tag problem. To address this, we have generated another line of transgenic mice that express wild-type human RTN3 under the control of an inducible promoter. Breeding this new line of mice with CaMK-tTA mice has now produced compound mice that will express the human RTN3 transgene. We have examined a couple of these new transgenic mice (eight months old) and found that RIDNs are also present in their hippocampus. Examination of elderly mouse brain has further confirmed that RIDNs are naturally occurring in their hippocampus (unpublished results). Hence, several lines of our studies all support that RTN3 is important in the formation of RIDNs.

    Biochemical studies showed that RTN3 tends to form dimers and high-molecular-weight RTN3 aggregates. A previous study also showed that RTN3 forms a dimer (Qi et al., 2003). Nogo (RTN4) has also been shown to form Nogo-dimers (Dodd et al., 2005). RTN proteins also form natural oligomers in the ER (Shibata et al., 2008). In addition, we found that transgenic mice expressing low levels of RTN3 transgene (line 1 and line 2) showed much less RIDN formation in their hippocampi (Hu et al., 2007). These results support that increased levels of RTN3 tend to form dimers, oligomers and aggregates, and should address Wataru Araki’s concerns. Our hypothesis that excessive aggregation of RTN3 promotes formation of RIDNs is supported by many data from biochemical, morphological, and animal model studies.

    References:

    Dodd DA, Niederoest B, Bloechlinger S, Dupuis L, Loeffler JP, Schwab ME. Nogo-A, -B, and -C are found on the cell surface and interact together in many different cell types. J Biol Chem. 2005 Apr 1;280(13):12494-502. PubMed.

    Hu X, Shi Q, Zhou X, He W, Yi H, Yin X, Gearing M, Levey A, Yan R. Transgenic mice overexpressing reticulon 3 develop neuritic abnormalities. EMBO J. 2007 Jun 6;26(11):2755-67. PubMed.

    Park JH, Gimbel DA, GrandPre T, Lee JK, Kim JE, Li W, Lee DH, Strittmatter SM. Alzheimer precursor protein interaction with the Nogo-66 receptor reduces amyloid-beta plaque deposition. J Neurosci. 2006 Feb 1;26(5):1386-95. PubMed.

    Qi B, Qi Y, Watari A, Yoshioka N, Inoue H, Minemoto Y, Yamashita K, Sasagawa T, Yutsudo M. Pro-apoptotic ASY/Nogo-B protein associates with ASYIP. J Cell Physiol. 2003 Aug;196(2):312-8. PubMed.

    Shibata Y, Voss C, Rist JM, Hu J, Rapoport TA, Prinz WA, Voeltz GK. The reticulon and DP1/Yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum. J Biol Chem. 2008 Jul 4;283(27):18892-904. PubMed.

    View all comments by Riqiang Yan

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References

News Citations

  1. BACE RNAi Scores in Mouse Model of AD

Paper Citations

  1. Bandtlow CE, Dlaska M, Pirker S, Czech T, Baumgartner C, Sperk G. Increased expression of Nogo-A in hippocampal neurons of patients with temporal lobe epilepsy. Eur J Neurosci. 2004 Jul;20(1):195-206. PubMed.
  2. Fergani A, Dupuis L, Jokic N, Larmet Y, de Tapia M, Rene F, Loeffler JP, Gonzalez de Aguilar JL. Reticulons as markers of neurological diseases: focus on amyotrophic lateral sclerosis. Neurodegener Dis. 2005;2(3-4):185-94. PubMed.
  3. Park JH, Gimbel DA, GrandPre T, Lee JK, Kim JE, Li W, Lee DH, Strittmatter SM. Alzheimer precursor protein interaction with the Nogo-66 receptor reduces amyloid-beta plaque deposition. J Neurosci. 2006 Feb 1;26(5):1386-95. PubMed.
  4. He W, Lu Y, Qahwash I, Hu XY, Chang A, Yan R. Reticulon family members modulate BACE1 activity and amyloid-beta peptide generation. Nat Med. 2004 Sep;10(9):959-65. Epub 2004 Aug 1 PubMed.
  5. Murayama KS, Kametani F, Saito S, Kume H, Akiyama H, Araki W. Reticulons RTN3 and RTN4-B/C interact with BACE1 and inhibit its ability to produce amyloid beta-protein. Eur J Neurosci. 2006 Sep;24(5):1237-44. PubMed.
  6. Hu X, Shi Q, Zhou X, He W, Yi H, Yin X, Gearing M, Levey A, Yan R. Transgenic mice overexpressing reticulon 3 develop neuritic abnormalities. EMBO J. 2007 Jun 6;26(11):2755-67. PubMed.
  7. Kume H, Konishi Y, Murayama KS, Kametani F, Araki W. Expression of reticulon 3 in Alzheimer's disease brain. Neuropathol Appl Neurobiol. 2009 Apr;35(2):178-88. PubMed.
  8. Shi Q, Hu X, Prior M, Yan R. The occurrence of aging-dependent reticulon 3 immunoreactive dystrophic neurites decreases cognitive function. J Neurosci. 2009 Apr 22;29(16):5108-15. PubMed.
  9. Borchelt DR, Ratovitski T, van Lare J, Lee MK, Gonzales V, Jenkins NA, Copeland NG, Price DL, Sisodia SS. Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron. 1997 Oct;19(4):939-45. PubMed.
  10. He W, Hu X, Shi Q, Zhou X, Lu Y, Fisher C, Yan R. Mapping of interaction domains mediating binding between BACE1 and RTN/Nogo proteins. J Mol Biol. 2006 Oct 27;363(3):625-34. PubMed.
  11. Singer O, Marr RA, Rockenstein E, Crews L, Coufal NG, Gage FH, Verma IM, Masliah E. Targeting BACE1 with siRNAs ameliorates Alzheimer disease neuropathology in a transgenic model. Nat Neurosci. 2005 Oct;8(10):1343-9. PubMed.
  12. McConlogue L, Buttini M, Anderson JP, Brigham EF, Chen KS, Freedman SB, Games D, Johnson-Wood K, Lee M, Zeller M, Liu W, Motter R, Sinha S. Partial reduction of BACE1 has dramatic effects on Alzheimer plaque and synaptic pathology in APP Transgenic Mice. J Biol Chem. 2007 Sep 7;282(36):26326-34. PubMed.

Further Reading

Papers

  1. Kume H, Murayama KS, Araki W. The two-hydrophobic domain tertiary structure of reticulon proteins is critical for modulation of beta-secretase BACE1. J Neurosci Res. 2009 Oct;87(13):2963-72. PubMed.
  2. Wojcik S, Engel WK, Yan R, McFerrin J, Askanas V. NOGO is increased and binds to BACE1 in sporadic inclusion-body myositis and in A beta PP-overexpressing cultured human muscle fibers. Acta Neuropathol. 2007 Nov;114(5):517-26. PubMed.
  3. Tesco G, Koh YH, Kang EL, Cameron AN, Das S, Sena-Esteves M, Hiltunen M, Yang SH, Zhong Z, Shen Y, Simpkins JW, Tanzi RE. Depletion of GGA3 stabilizes BACE and enhances beta-secretase activity. Neuron. 2007 Jun 7;54(5):721-37. PubMed.
  4. Gil V, Nicolas O, Mingorance A, Ureña JM, Tang BL, Hirata T, Sáez-Valero J, Ferrer I, Soriano E, Del Río JA. Nogo-A expression in the human hippocampus in normal aging and in Alzheimer disease. J Neuropathol Exp Neurol. 2006 May;65(5):433-44. PubMed.
  5. Yan R, Shi Q, Hu X, Zhou X. Reticulon proteins: emerging players in neurodegenerative diseases. Cell Mol Life Sci. 2006 Apr;63(7-8):877-89. PubMed.
  6. Laird FM, Cai H, Savonenko AV, Farah MH, He K, Melnikova T, Wen H, Chiang HC, Xu G, Koliatsos VE, Borchelt DR, Price DL, Lee HK, Wong PC. BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions. J Neurosci. 2005 Dec 14;25(50):11693-709. PubMed.

Primary Papers

  1. Shi Q, Prior M, He W, Tang X, Hu X, Yan R. Reduced amyloid deposition in mice overexpressing RTN3 is adversely affected by preformed dystrophic neurites. J Neurosci. 2009 Jul 22;29(29):9163-73. PubMed.

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