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.
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 neuritis. J Neurosci. 2009 July 22;29(29):9163-9173. Abstract
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