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Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice.

Wang J, Wang CE, Orr A, Tydlacka S, Li SH, Li XJ - J. Cell Biol. (2008)

Bottom Line: In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function.We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons.Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.

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Expression of synaptic UPS reporters in neurons. (A) Schematic map of DNA constructs for expressing fluorescent UPS reporters. RFP or GFPu is added to the C terminus of PSD95 or SNAP25 in an adenoviral vector to express fusion proteins under the control of the human synapsin 1 promoter. (B) Western blots showing the expression of these fusion proteins in HEK293 cells after adenoviral infection. (C) Expression of fluorescent UPS reporters in primary cultured cortical neurons after infection with adenoviral SNAP25-RFP vector. SNAP25-RFP was expressed in cultured primary neurons (NeuN-positive) but not in cultured glial cells (GFAP-positive). (D) In the mouse brain striatum injected with adenoviral SNAP25-RFP reporter, SNAP25-RFP was also expressed in neurons, but not glial cells. Immunostaining for GFAP or NeuN (green), nuclear staining (blue) by Hoechst, and SNAP25-RFP (red) were revealed by fluorescent microscopy. Bars, 5 μm (C) and 10 μm (D).
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fig1: Expression of synaptic UPS reporters in neurons. (A) Schematic map of DNA constructs for expressing fluorescent UPS reporters. RFP or GFPu is added to the C terminus of PSD95 or SNAP25 in an adenoviral vector to express fusion proteins under the control of the human synapsin 1 promoter. (B) Western blots showing the expression of these fusion proteins in HEK293 cells after adenoviral infection. (C) Expression of fluorescent UPS reporters in primary cultured cortical neurons after infection with adenoviral SNAP25-RFP vector. SNAP25-RFP was expressed in cultured primary neurons (NeuN-positive) but not in cultured glial cells (GFAP-positive). (D) In the mouse brain striatum injected with adenoviral SNAP25-RFP reporter, SNAP25-RFP was also expressed in neurons, but not glial cells. Immunostaining for GFAP or NeuN (green), nuclear staining (blue) by Hoechst, and SNAP25-RFP (red) were revealed by fluorescent microscopy. Bars, 5 μm (C) and 10 μm (D).

Mentions: The fluorescent UPS reporter GFPu, which is a green fluorescent fusion protein tagged with a CL-1 degron sequence (16 amino acids) specific for ubiquitination and degradation by the proteasome, has been widely used to examine the activity of the proteasome in cells (Bence et al., 2001; Dong et al., 2004; Avraham et al., 2005; Bennett et al., 2005). To specifically measure synaptic UPS activity using this reporter, we fused the postsynaptic protein PSD95 or the presynaptic protein SNAP25 to GFPu in order to target this reporter to postsynaptic or presynaptic terminals. To ensure neuronal expression, the fusion proteins are expressed via an adenoviral vector under the control of the neuronal promoter for human synapsin 1, a synaptic vesicle-associated protein (Fig. 1 A). As controls, we also generated cDNAs encoding for DsRed fusion proteins (RFP), which provide red fluorescent signals but are not degraded rapidly by the UPS. Thus, the ratio of GFPu to RFP (GFPu/RFP) reflects the relative activity of the UPS when GFPu and RFP reporters are coexpressed in the same cells.


Impaired ubiquitin-proteasome system activity in the synapses of Huntington's disease mice.

Wang J, Wang CE, Orr A, Tydlacka S, Li SH, Li XJ - J. Cell Biol. (2008)

Expression of synaptic UPS reporters in neurons. (A) Schematic map of DNA constructs for expressing fluorescent UPS reporters. RFP or GFPu is added to the C terminus of PSD95 or SNAP25 in an adenoviral vector to express fusion proteins under the control of the human synapsin 1 promoter. (B) Western blots showing the expression of these fusion proteins in HEK293 cells after adenoviral infection. (C) Expression of fluorescent UPS reporters in primary cultured cortical neurons after infection with adenoviral SNAP25-RFP vector. SNAP25-RFP was expressed in cultured primary neurons (NeuN-positive) but not in cultured glial cells (GFAP-positive). (D) In the mouse brain striatum injected with adenoviral SNAP25-RFP reporter, SNAP25-RFP was also expressed in neurons, but not glial cells. Immunostaining for GFAP or NeuN (green), nuclear staining (blue) by Hoechst, and SNAP25-RFP (red) were revealed by fluorescent microscopy. Bars, 5 μm (C) and 10 μm (D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2290845&req=5

fig1: Expression of synaptic UPS reporters in neurons. (A) Schematic map of DNA constructs for expressing fluorescent UPS reporters. RFP or GFPu is added to the C terminus of PSD95 or SNAP25 in an adenoviral vector to express fusion proteins under the control of the human synapsin 1 promoter. (B) Western blots showing the expression of these fusion proteins in HEK293 cells after adenoviral infection. (C) Expression of fluorescent UPS reporters in primary cultured cortical neurons after infection with adenoviral SNAP25-RFP vector. SNAP25-RFP was expressed in cultured primary neurons (NeuN-positive) but not in cultured glial cells (GFAP-positive). (D) In the mouse brain striatum injected with adenoviral SNAP25-RFP reporter, SNAP25-RFP was also expressed in neurons, but not glial cells. Immunostaining for GFAP or NeuN (green), nuclear staining (blue) by Hoechst, and SNAP25-RFP (red) were revealed by fluorescent microscopy. Bars, 5 μm (C) and 10 μm (D).
Mentions: The fluorescent UPS reporter GFPu, which is a green fluorescent fusion protein tagged with a CL-1 degron sequence (16 amino acids) specific for ubiquitination and degradation by the proteasome, has been widely used to examine the activity of the proteasome in cells (Bence et al., 2001; Dong et al., 2004; Avraham et al., 2005; Bennett et al., 2005). To specifically measure synaptic UPS activity using this reporter, we fused the postsynaptic protein PSD95 or the presynaptic protein SNAP25 to GFPu in order to target this reporter to postsynaptic or presynaptic terminals. To ensure neuronal expression, the fusion proteins are expressed via an adenoviral vector under the control of the neuronal promoter for human synapsin 1, a synaptic vesicle-associated protein (Fig. 1 A). As controls, we also generated cDNAs encoding for DsRed fusion proteins (RFP), which provide red fluorescent signals but are not degraded rapidly by the UPS. Thus, the ratio of GFPu to RFP (GFPu/RFP) reflects the relative activity of the UPS when GFPu and RFP reporters are coexpressed in the same cells.

Bottom Line: In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function.We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons.Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT
Huntington's disease (HD) is caused by the expansion of a polyglutamine tract in the N-terminal region of huntingtin (htt) and is characterized by selective neurodegeneration. In addition to forming nuclear aggregates, mutant htt accumulates in neuronal processes as well as synapses and affects synaptic function. However, the mechanism for the synaptic toxicity of mutant htt remains to be investigated. We targeted fluorescent reporters for the ubiquitin-proteasome system (UPS) to presynaptic or postsynaptic terminals of neurons. Using these reporters and biochemical assays of isolated synaptosomes, we found that mutant htt decreases synaptic UPS activity in cultured neurons and in HD mouse brains that express N-terminal or full-length mutant htt. Given that the UPS is a key regulator of synaptic plasticity and function, our findings offer insight into the selective neuronal dysfunction seen in HD and also establish a method to measure synaptic UPS activity in other neurological disease models.

Show MeSH
Related in: MedlinePlus