Dynactin 6 deficiency enhances aging-associated dystrophic neurite formation in mouse brains
Introduction
The presence of dystrophic neurites (DNs) is one of distinguishing features in brains of Alzheimer's disease (AD) patients and a contributing factor to synaptic dysfunction (Hu et al., 2007; Joachim et al., 1989; Lenders et al., 1989). In AD brains, various proteins and organelles accumulate in DNs surrounding amyloid beta (Aβ) plaques (Dickson et al., 1999; Nixon et al., 2005; Sharoar et al., 2019). Reticulon 3 (RTN3), a tubular endoplasmic reticulum (ER)-shaping protein, was previously shown to mark an abundant population of DNs clustered around Aβ plaque in AD brains, and therefore named RTN3-immunoreactive DNs (RIDNs) (Hu et al., 2007; Shi et al., 2013).
Besides the clustered form of DNs surrounding Aβ plaques in AD brains, disperse RIDNs also emerge throughout the hippocampus CA1 region in aged wild-type (WT) mouse brains. Furthermore, the formation of disperse RIDNs is accelerated when RTN3 is overexpressed in transgenic RTN3 (Tg-RTN3) mouse brains (Hu et al., 2007; Shi et al., 2009). The presence of RIDNs impairs learning and memory in Tg-RTN3 mice, as determined by electrophysiological recordings and behavioral tests (Hu et al., 2007; Shi et al., 2013).
RTN3 is the neuronal-enriched member of the RTN family (with four members: RTN1 to RTN4), which is localized in the tubular domain of ER that controls the curvature of the ER membrane (Voeltz et al., 2006). All RTNs have a common C-terminal homology domain, known as an RTN homology domain (RHD), and a diversified N-terminal domain (Oertle and Schwab, 2003). 2 transmembrane (TM) domains, TM1 and TM2, of RTNs are localized within the RHD and play critical roles in ER tubulation by membrane embedding via interaction with other ER tubule proteins such as the receptor expression-enhancing proteins (REEPs) (Sharoar et al., 2016; Sharoar and Yan, 2017; Voeltz et al., 2006). Interestingly, although RTN1 and RTN4 are also expressed by neurons, these 2 RTN family members are not enriched in RIDNs (Hu et al., 2007; Shi et al., 2017), suggesting no direct roles in the formation of DNs. RIDNs appear to interact with other tubular ER-shaping proteins, REEP2 and REEP5, in AD brains and in Tg-RTN3 mouse brains, and could have an age-dependent accumulation with tubular ER at axonal termini (Sharoar et al., 2016). Furthermore, RTN3-mediated tubular ER clustering also induces mitochondrial degeneration. Hence, RTN3 appears to be a key molecule for RIDN formation in aging mouse brains because REEP2/5-marked RIDNs are absent in older RTN3 knockout mouse brains (Sharoar et al., 2016).
Biochemically, RTN3 can form dimers and multimers, and high molecular weight (HMW)-RTN3 aggregates are often seen in the hippocampal area of brains (Hu et al., 2007; Shibata et al., 2008). The degree of HMW-RTN3 aggregate formation in Tg-RTN3 mouse hippocampi and in AD brains corresponds to the formation of dispersed and clustered RIDNs, respectively (Hu et al., 2007; Shi et al., 2009). In AD brains, Aβ-induced trafficking impairment and a defective proteasomal system have been suggested to stimulate the formation of HMW-RTN3 aggregates and clustered RIDNs surrounding Aβ plaques (Prior et al., 2010). However, how RTN3 aggregation and RIDN formation specifically occurs in the hippocampal region during aging in mouse brains is not yet clear.
By utilizing yeast 2-hybrid screening, we identified a subunit of dynein transport machinery complex, dynactin 6 (DCTN6), that interacts with the N-terminal domain of RTN3. We found that DCTN6 protein levels were reduced during aging in the hippocampus compared to the cortex in WT mouse brains. Knocking down DCTN6 enhanced RTN3 protein levels and the spontaneous formation of dispersed RIDNs in mouse brains. Although AAV-mediated overexpression of DCTN6 failed to attenuate RIDN formation in mouse brains, we found that DCTN6-RTN3 interactions altered tubular ER trafficking in axons. DCTN6 deficiency in the hippocampus impaired axonal transport of RTN3, and this impairment aggravated RTN3 aggregation, tubular ER accumulation, and RIDN formation in the aging mouse brain. Hence, DCTN6 appears to a regulator of RTN3 axonal transport, and worth further investigation to uncover its physiological functions.
Section snippets
Mouse strains
Tg-RTN3 mice were generated and genotyping was performed as described previously (Hu et al., 2007). DCTN6 heterozygous mutant mice (DCTN6+/-) were purchased from Jackson Laboratory (stock # 029000) and the genotyping was performed according to vendor instructions. Both Tg-RTN3 and DCTN6+/- strains were maintained by breeding with B6 mice (Jackson Lab stock # 000664). All mice in the study were maintained and used according to protocols approved by the Institutional Animal Care and Use Committee
DCTN6 interacts with the N-terminal domain of RTN3
To understand how RTN3 mediates RIDN formation in the hippocampus, we speculated that an RTN3-interacting protein in the hippocampus facilitates this formation. We utilized a well-established yeast two-hybrid system to screen for RTN3-interacting partners in a human brain cDNA library that contains ∼1 million clones. Our screen using N-terminal residues (amino acids 1-61) of the RTN3 protein as bait identified ∼300 positive clones, and these were PCR amplified. Among the 300 clones, we obtained
Discussion
The formation of DNs is a distinguishing feature of AD brains because a diffused form of Aβ plaques is also present in cognitively normal elder human brains that lack DNs (Dickson et al., 1999; Fukumoto et al., 1996; Joachim et al., 1989; Lenders et al., 1989). RIDNs comprise a major proportion of DNs in AD brains compared to other types of DNs that are detected by tau, ubiquitin, or neurofilaments (Dickson et al., 1999; Hu et al., 2007; Lenders et al., 1989). Hippocampus-specific disperse RIDN
Author contribution
MGS deigned the experiment, performed most of the experiments, wrote this manuscript. JZ, MB and WH performed some experiments. RY mentored the experiment and critically edited this manuscript.
Disclosure statement
The authors declare no conflict of interest.
Acknowledgements
We thank Dr. Christopher Bonin and Dr. Geneva Hargis for critical reading of this manuscript. This work is supported by an award from Alzheimer's Association to (AARF-17-504724) MGS and NIH grants to RY (AG025493 and RFA046929).
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