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Viral expression of ALS-linked ubiquilin-2 mutants causes inclusion pathology and behavioral deficits in mice.

Ceballos-Diaz C, Rosario AM, Park HJ, Chakrabarty P, Sacino A, Cruz PE, Siemienski Z, Lara N, Moran C, Ravelo N, Golde TE, McFarland NR - Mol Neurodegener (2015)

Bottom Line: In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates.In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil.Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, 1275 Center Dr, PO Box 100159, Gainesville, FL, 32610, USA.

ABSTRACT

Background: UBQLN2 mutations have recently been associated with familial forms of amyotrophic lateral sclerosis (ALS) and ALS-dementia. UBQLN2 encodes for ubiquilin-2, a member of the ubiquitin-like protein family which facilitates delivery of ubiquitinated proteins to the proteasome for degradation. To study the potential role of ubiquilin-2 in ALS, we used recombinant adeno-associated viral (rAAV) vectors to express UBQLN2 and three of the identified ALS-linked mutants (P497H, P497S, and P506T) in primary neuroglial cultures and in developing neonatal mouse brains.

Results: In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates. Intracerebroventricular injection of FVB mice at post-natal day 0 with rAAV2/8 expressing wild type or mutant UBQLN2 resulted in widespread, sustained expression of ubiquilin-2 in brain. In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil. Ubiquilin-2 inclusions co-localized with ubiquitin, p62/SQSTM, optineurin, and occasionally TDP-43, but were negative for α-synuclein, neurofilament, tau, and FUS. Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates. Mice expressing mutant forms of UBQLN2 variably developed a motor phenotype at 3-4 months, including nonspecific clasping and rotarod deficits.

Conclusions: These findings demonstrate that UBQLN2 mutants (P497H, P497S, and P506T) induce proteinopathy and cause behavioral deficits, supporting a "toxic" gain-of-function, which may contribute to ALS pathology. These data establish also that our rAAV model can be used to rapidly assess the pathological consequences of various UBQLN2 mutations and provides an agile system to further interrogate the molecular mechanisms of ubiquilins in neurodegeneration.

No MeSH data available.


Related in: MedlinePlus

Mutant Ubiquilin-2 overexpression results in punctate intracellular accumulations in primary mixed neuroglia cultures. a. Cells transduced with AAV-UBQLN2(WT) show ubiquilin-2 immunoreactivity (green) diffusely present in the cytoplasm and cell processes with few small punctate accumulations. In contrast, UBQLN2 mutants (P497S, P497H, and P506T) result in large intracellular ubiquilin-2 accumulations both in neuronal soma and processes (red, labelled with MAP2). Ubiquilin-2 accumulations in processes have a “bead on a string”-like appearance particularly for P497H and P506T mutants. b. Some ubiquilin-2 accumulations (green) are outside of neurons and colocalized with astrocytes in culture, labeled with GFAP-imunoreactivity (red). c. Western blot of TX-soluble and insoluble fractions show that all UBQLN2 mutants and not WT accumulate in the TX-insoluble/SDS fraction, suggesting formation insoluble aggregates. d. Graph of d2EGFP signal normalized to actin in HEK293 cells transfected with WT and mutant ubiquilin-2. Both P497S and P506T mutants show impaired proteasomal degradation of the d2EGFP reporter compared to the P497H mutant and WT ubiquilin-2. *p < 0.05, **p < 0.01
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Fig1: Mutant Ubiquilin-2 overexpression results in punctate intracellular accumulations in primary mixed neuroglia cultures. a. Cells transduced with AAV-UBQLN2(WT) show ubiquilin-2 immunoreactivity (green) diffusely present in the cytoplasm and cell processes with few small punctate accumulations. In contrast, UBQLN2 mutants (P497S, P497H, and P506T) result in large intracellular ubiquilin-2 accumulations both in neuronal soma and processes (red, labelled with MAP2). Ubiquilin-2 accumulations in processes have a “bead on a string”-like appearance particularly for P497H and P506T mutants. b. Some ubiquilin-2 accumulations (green) are outside of neurons and colocalized with astrocytes in culture, labeled with GFAP-imunoreactivity (red). c. Western blot of TX-soluble and insoluble fractions show that all UBQLN2 mutants and not WT accumulate in the TX-insoluble/SDS fraction, suggesting formation insoluble aggregates. d. Graph of d2EGFP signal normalized to actin in HEK293 cells transfected with WT and mutant ubiquilin-2. Both P497S and P506T mutants show impaired proteasomal degradation of the d2EGFP reporter compared to the P497H mutant and WT ubiquilin-2. *p < 0.05, **p < 0.01

Mentions: Recombinant AAV2/8 expressing ubiquilin-2 WT or ALS-linked mutants (P497S, P497H and P506T) was used to transduce primary neuroglial cultures at DIV + 6. Four days post-transduction cells were analyzed by immunofluorescence and biochemistry. Neurons were identified by MAP2 and astrocytes by GFAP co-immunostaining. Ubiquilin-2 expression was primarily observed in neurons in E16 cultures, but also seen in some astrocytes. In cells expressing ubiquilin-2 WT or pathologic mutants, there was low level of diffuse ubiquilin-2 immunoreactivity throughout the neuronal perikarya. Most notably, large accumulations of ubiquilin-2 were seen in both the neuronal cytoplasm and processes. Intracellular ubiquilin-2-postive accumulations, although present in cultures transduced with AAV-UBQLN2(WT), were larger and more prevalent in cultures transduced with mutant UBQLN2 (Fig. 1a). Also, neurons transduced with either P497S, P497H or P506T mutant ubiquilin-2 displayed frequent ubiquilin-2-postive punctate accumulations in neuronal processes with a “bead on a string”-like appearance, suggesting an altered subcellular distribution. These puncta were more apparent in cultures transduced with the P497H and P506T UBQLN2 mutants and associated with apparent dystrophic changes in neurites. Some ubiquilin-2-postive accumulations were located outside neurons and colocalized with the astrocytic marker GFAP, but not the microglial marker Iba-1 (Fig. 1b). Preliminary screen to identify subcellular localization of intracellular ubiquilin-2 accumulations revealed no colocalization with early endosomal markers such as EEA1 or Rab5; late endosomes, Rab7; autophagosomes, LC3; or lysosomes, LAMP1 (data not shown).Fig. 1


Viral expression of ALS-linked ubiquilin-2 mutants causes inclusion pathology and behavioral deficits in mice.

Ceballos-Diaz C, Rosario AM, Park HJ, Chakrabarty P, Sacino A, Cruz PE, Siemienski Z, Lara N, Moran C, Ravelo N, Golde TE, McFarland NR - Mol Neurodegener (2015)

Mutant Ubiquilin-2 overexpression results in punctate intracellular accumulations in primary mixed neuroglia cultures. a. Cells transduced with AAV-UBQLN2(WT) show ubiquilin-2 immunoreactivity (green) diffusely present in the cytoplasm and cell processes with few small punctate accumulations. In contrast, UBQLN2 mutants (P497S, P497H, and P506T) result in large intracellular ubiquilin-2 accumulations both in neuronal soma and processes (red, labelled with MAP2). Ubiquilin-2 accumulations in processes have a “bead on a string”-like appearance particularly for P497H and P506T mutants. b. Some ubiquilin-2 accumulations (green) are outside of neurons and colocalized with astrocytes in culture, labeled with GFAP-imunoreactivity (red). c. Western blot of TX-soluble and insoluble fractions show that all UBQLN2 mutants and not WT accumulate in the TX-insoluble/SDS fraction, suggesting formation insoluble aggregates. d. Graph of d2EGFP signal normalized to actin in HEK293 cells transfected with WT and mutant ubiquilin-2. Both P497S and P506T mutants show impaired proteasomal degradation of the d2EGFP reporter compared to the P497H mutant and WT ubiquilin-2. *p < 0.05, **p < 0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495639&req=5

Fig1: Mutant Ubiquilin-2 overexpression results in punctate intracellular accumulations in primary mixed neuroglia cultures. a. Cells transduced with AAV-UBQLN2(WT) show ubiquilin-2 immunoreactivity (green) diffusely present in the cytoplasm and cell processes with few small punctate accumulations. In contrast, UBQLN2 mutants (P497S, P497H, and P506T) result in large intracellular ubiquilin-2 accumulations both in neuronal soma and processes (red, labelled with MAP2). Ubiquilin-2 accumulations in processes have a “bead on a string”-like appearance particularly for P497H and P506T mutants. b. Some ubiquilin-2 accumulations (green) are outside of neurons and colocalized with astrocytes in culture, labeled with GFAP-imunoreactivity (red). c. Western blot of TX-soluble and insoluble fractions show that all UBQLN2 mutants and not WT accumulate in the TX-insoluble/SDS fraction, suggesting formation insoluble aggregates. d. Graph of d2EGFP signal normalized to actin in HEK293 cells transfected with WT and mutant ubiquilin-2. Both P497S and P506T mutants show impaired proteasomal degradation of the d2EGFP reporter compared to the P497H mutant and WT ubiquilin-2. *p < 0.05, **p < 0.01
Mentions: Recombinant AAV2/8 expressing ubiquilin-2 WT or ALS-linked mutants (P497S, P497H and P506T) was used to transduce primary neuroglial cultures at DIV + 6. Four days post-transduction cells were analyzed by immunofluorescence and biochemistry. Neurons were identified by MAP2 and astrocytes by GFAP co-immunostaining. Ubiquilin-2 expression was primarily observed in neurons in E16 cultures, but also seen in some astrocytes. In cells expressing ubiquilin-2 WT or pathologic mutants, there was low level of diffuse ubiquilin-2 immunoreactivity throughout the neuronal perikarya. Most notably, large accumulations of ubiquilin-2 were seen in both the neuronal cytoplasm and processes. Intracellular ubiquilin-2-postive accumulations, although present in cultures transduced with AAV-UBQLN2(WT), were larger and more prevalent in cultures transduced with mutant UBQLN2 (Fig. 1a). Also, neurons transduced with either P497S, P497H or P506T mutant ubiquilin-2 displayed frequent ubiquilin-2-postive punctate accumulations in neuronal processes with a “bead on a string”-like appearance, suggesting an altered subcellular distribution. These puncta were more apparent in cultures transduced with the P497H and P506T UBQLN2 mutants and associated with apparent dystrophic changes in neurites. Some ubiquilin-2-postive accumulations were located outside neurons and colocalized with the astrocytic marker GFAP, but not the microglial marker Iba-1 (Fig. 1b). Preliminary screen to identify subcellular localization of intracellular ubiquilin-2 accumulations revealed no colocalization with early endosomal markers such as EEA1 or Rab5; late endosomes, Rab7; autophagosomes, LC3; or lysosomes, LAMP1 (data not shown).Fig. 1

Bottom Line: In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates.In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil.Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, 1275 Center Dr, PO Box 100159, Gainesville, FL, 32610, USA.

ABSTRACT

Background: UBQLN2 mutations have recently been associated with familial forms of amyotrophic lateral sclerosis (ALS) and ALS-dementia. UBQLN2 encodes for ubiquilin-2, a member of the ubiquitin-like protein family which facilitates delivery of ubiquitinated proteins to the proteasome for degradation. To study the potential role of ubiquilin-2 in ALS, we used recombinant adeno-associated viral (rAAV) vectors to express UBQLN2 and three of the identified ALS-linked mutants (P497H, P497S, and P506T) in primary neuroglial cultures and in developing neonatal mouse brains.

Results: In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates. Intracerebroventricular injection of FVB mice at post-natal day 0 with rAAV2/8 expressing wild type or mutant UBQLN2 resulted in widespread, sustained expression of ubiquilin-2 in brain. In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil. Ubiquilin-2 inclusions co-localized with ubiquitin, p62/SQSTM, optineurin, and occasionally TDP-43, but were negative for α-synuclein, neurofilament, tau, and FUS. Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates. Mice expressing mutant forms of UBQLN2 variably developed a motor phenotype at 3-4 months, including nonspecific clasping and rotarod deficits.

Conclusions: These findings demonstrate that UBQLN2 mutants (P497H, P497S, and P506T) induce proteinopathy and cause behavioral deficits, supporting a "toxic" gain-of-function, which may contribute to ALS pathology. These data establish also that our rAAV model can be used to rapidly assess the pathological consequences of various UBQLN2 mutations and provides an agile system to further interrogate the molecular mechanisms of ubiquilins in neurodegeneration.

No MeSH data available.


Related in: MedlinePlus