Cellular and Molecular Neurobiology

, Volume 36, Issue 3, pp 459–470 | Cite as

Potential Transfer of Polyglutamine and CAG-Repeat RNA in Extracellular Vesicles in Huntington’s Disease: Background and Evaluation in Cell Culture

  • Xuan Zhang
  • Erik R. Abels
  • Jasmina S. Redzic
  • Julia Margulis
  • Steve Finkbeiner
  • Xandra O. Breakefield
Review Paper

Abstract

In Huntington’s disease (HD) the imperfect expanded CAG repeat in the first exon of the HTT gene leads to the generation of a polyglutamine (polyQ) protein, which has some neuronal toxicity, potentially mollified by formation of aggregates. Accumulated research, reviewed here, implicates both the polyQ protein and the expanded repeat RNA in causing toxicity leading to neurodegeneration in HD. Different theories have emerged as to how the neurodegeneration spreads throughout the brain, with one possibility being the transport of toxic protein and RNA in extracellular vesicles (EVs). Most cell types in the brain release EVs and these have been shown to contain neurodegenerative proteins in the case of prion protein and amyloid-beta peptide. In this study, we used a model culture system with an overexpression of HTT-exon 1 polyQ-GFP constructs in human 293T cells and found that the EVs did incorporate both the polyQ-GFP protein and expanded repeat RNA. Striatal mouse neural cells were able to take up these EVs with a consequent increase in the green fluorescent protein (GFP) and polyQ-GFP RNAs, but with no evidence of uptake of polyQ-GFP protein or any apparent toxicity, at least over a relatively short period of exposure. A differentiated striatal cell line expressing endogenous levels of Hdh mRNA containing the expanded repeat incorporated more of this mRNA into EVs as compared to similar cells expressing this mRNA with a normal repeat length. These findings support the potential of EVs to deliver toxic expanded trinucleotide repeat RNAs from one cell to another, but further work will be needed to evaluate potential EV and cell-type specificity of transfer and effects of long-term exposure. It seems likely that expanded HD-associated repeat RNA may appear in biofluids and may have use as biomarkers of disease state and response to therapy.

Keywords

Exosomes Trinucleotide repeat Neurodegeneration Huntington’s disease 

Abbreviations

CSF

Cerebral spinal fluid

DMEM

Dulbecco’s modified Eagle’s medium

EVs

Extracellular vesicles

FBS

Fetal bovine serum

FGF

Fibroblast growth factor

GFP

Green fluorescent protein

HRP

Horseradish peroxide

IBMX

Isobutyl-1-methylxanthine

M.O.I.

Multiplicity of infection

palm

Palmitoylated

PEI

Polyethylenimine

polyQ

Polyglutamine

Notes

Acknowledgments

We thank Ms. Suzanne McDavitt for skilled editorial assistance. Drs. Marian DiFiglia, Ellen Sapp, and Neal Aronin for insightful comments on HD pathophysiology. This work was supported by the NIH Common Fund through the Office of Strategic Coordination/Office of the NIH Director, NCI U19 CA179563 (XOB), and NIH NRSA NIA postdoctoral training grant, 2T32AG000222 (JSR). Additional support was from NIH 3R01 NS039074 (SF). Lentiviral vectors were produced by the MGH Vector Core supported by NIN/NINDS P30 NS045775 (XOB and Dr. Bakhos Tannous).

Supplementary material

10571_2016_350_MOESM1_ESM.tiff (11.4 mb)
RT-PCR of polyQ-GFP RNA with different repeat lengths from transduced 293T cells and EVs. 293T cells were transduced using lentiviral vectors encoding GFP (not shown), Httex1-25Q-GFP, and Httex1-97Q-GFP. Two weeks later (equivalent to about 6 passages) RNA from cells and EVs was extracted using the miRNeasy mini kit. RT-PCR products were analyzed on 1 % agarose gel using 1 kb Quick-Load ladder. [Open arrowhead—25Q-GFP; solid arrowhead—97Q-GFP; gray arrowhead—GFP RNAs.] (TIFF 11720 kb)
10571_2016_350_MOESM2_ESM.tiff (11.4 mb)
Bright field image showing striatal neuron-like STHdhQ7/Q7 and STHdhQ111/Q111 cells 12 h after differentiation. Immortalized mouse striatal cell lines, STHdhQ111/Q111 and STHdhQ7/Q7 were normally cultured in high glucose DMEM (Corning) plus 10 % FBS and 40 mg/ml of G418 at 33oC. After neuronal differentiation, which was induced by incubation with a dopamine cocktail of α-FGF (10 ng/ml), 3-IBMX (240 μM), forskolin (48.6 μM), and dopamine (5 μM) (Sigma) in DMEM/F12 for 12 h, cells develop long neuron-like processes with STHdhQ7/Q7 appearing to have larger soma than the STHdh Q111/Q111 cells. (TIFF 11720 kb)
10571_2016_350_MOESM3_ESM.tiff (11.4 mb)
HTT protein was detected in the STHdh111/111 cells but not in the EVs from these cells. Striatal cells were allowed to attach for 6 h before being differentiated. Cells and EVs in conditioned media were harvested from both striatal cell lines 48 h after differentiation. Western blots were performed for both striatal cells and EV lysates using monoclonal 3B5H10 antibody. The HTT protein (approx. 350 kDa) was found in STHdh111/111 cells, but was not detectable in STHdh7/7 cells, consistent with the low levels of HTT protein in the latter (Krauss et al. 2013). No HTT immunoreactive proteins were found in the EVs from these cell lines. Open circle = non-specific protein; solid circle = HTT protein. (TIFF 11720 kb)
10571_2016_350_MOESM4_ESM.docx (54 kb)
Supplementary material 4 (DOCX 53 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Xuan Zhang
    • 1
    • 2
    • 3
  • Erik R. Abels
    • 1
    • 2
    • 3
  • Jasmina S. Redzic
    • 4
  • Julia Margulis
    • 5
  • Steve Finkbeiner
    • 5
  • Xandra O. Breakefield
    • 1
    • 2
    • 3
  1. 1.Molecular Neurogenetics Unit, Department of NeurologyMassachusetts General Hospital-EastCharlestownUSA
  2. 2.Center for Molecular Imaging Research, Department of RadiologyMassachusetts General HospitalBostonUSA
  3. 3.Center for NeuroDiscoveryHarvard Medical SchoolBostonUSA
  4. 4.Department of Pharmaceutical SciencesUniversity of Colorado Denver Skaggs School of Pharmacy and Pharmaceutical SciencesAuroraUSA
  5. 5.Gladstone Institute of Neurological Disease and the Departments of Neurology and PhysiologyUniversity of California San FranciscoSan FranciscoUSA

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