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Mutant HSPB8 causes motor neuron-specific neurite degeneration.

Irobi J, Almeida-Souza L, Asselbergh B, De Winter V, Goethals S, Dierick I, Krishnan J, Timmermans JP, Robberecht W, De Jonghe P, Van Den Bosch L, Janssens S, Timmerman V - Hum. Mol. Genet. (2010)

Bottom Line: Furthermore, expression of the K141E (and to a lesser extent, K141N) mutation also induced spheroids in the neurites.While overt in motor neurons, these phenotypes were only very mildly present in sensory neurons and completely absent in cortical neurons.Also glial cells did not show an altered phenotype upon expression of mutant HSPB8.

View Article: PubMed Central - PubMed

Affiliation: Peripheral Neuropathy, VIB Department of Molecular Genetics, University of Antwerp, Antwerp, Belgium.

ABSTRACT
Missense mutations (K141N and K141E) in the alpha-crystallin domain of the small heat shock protein HSPB8 (HSP22) cause distal hereditary motor neuropathy (distal HMN) or Charcot-Marie-Tooth neuropathy type 2L (CMT2L). The mechanism through which mutant HSPB8 leads to a specific motor neuron disease phenotype is currently unknown. To address this question, we compared the effect of mutant HSPB8 in primary neuronal and glial cell cultures. In motor neurons, expression of both HSPB8 K141N and K141E mutations clearly resulted in neurite degeneration, as manifested by a reduction in number of neurites per cell, as well as in a reduction in average length of the neurites. Furthermore, expression of the K141E (and to a lesser extent, K141N) mutation also induced spheroids in the neurites. We did not detect any signs of apoptosis in motor neurons, showing that mutant HSPB8 resulted in neurite degeneration without inducing neuronal death. While overt in motor neurons, these phenotypes were only very mildly present in sensory neurons and completely absent in cortical neurons. Also glial cells did not show an altered phenotype upon expression of mutant HSPB8. These findings show that despite the ubiquitous presence of HSPB8, only motor neurons appear to be affected by the K141N and K141E mutations which explain the predominant motor neuron phenotype in distal HMN and CMT2L.

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Mutant HSPB8 induces neurite degeneration in a small proportion of primary sensory neurons. Mouse sensory neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV6 and immunostained at DIV10 using β-III tubulin antibody. Merged confocal micrographs of GFP (green) and β-III tubulin (red) are shown (A–D). No signs of neurite degeneration were found in sensory neurons expressing GFP (A), HSPB8-WT (B) and HSPB8-K141N (C), while the neurites of some cells expressing HSPB8-K141E were clearly beaded (arrow) (D). Scale bar = 10 µm. The incidence of neurite degeneration was quantified by counting the proportion of neurons with beaded neurites (E). **P-value < 0.01.
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DDQ234F4: Mutant HSPB8 induces neurite degeneration in a small proportion of primary sensory neurons. Mouse sensory neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV6 and immunostained at DIV10 using β-III tubulin antibody. Merged confocal micrographs of GFP (green) and β-III tubulin (red) are shown (A–D). No signs of neurite degeneration were found in sensory neurons expressing GFP (A), HSPB8-WT (B) and HSPB8-K141N (C), while the neurites of some cells expressing HSPB8-K141E were clearly beaded (arrow) (D). Scale bar = 10 µm. The incidence of neurite degeneration was quantified by counting the proportion of neurons with beaded neurites (E). **P-value < 0.01.

Mentions: The majority of the primary sensory neurons expressing mutant K141N-HSPB8 appeared healthy and morphologically similar to WT-HSPB8 or native GFP expressing sensory neurons (Fig. 4A–C). However, beaded neurites were observed in ∼10% of the mutant K141E-HSPB8 expressing sensory neuronal cells, similarly to what we saw before in motor neurons, though to a lower extent (10 versus 80%) (Fig. 4D) (GFP = 0.0 ± 0.0%, n = 655; WT-HSPB8-GFP = 0.0 ± 0.0%, n = 436; K141N-GFP = 0.5 ± 0.0%, n = 578, P-value = 0.221 and K141E-GFP = 9.7 ± 1.9%, n = 660, P-value = 0.000) (Fig. 4E).Figure 4.


Mutant HSPB8 causes motor neuron-specific neurite degeneration.

Irobi J, Almeida-Souza L, Asselbergh B, De Winter V, Goethals S, Dierick I, Krishnan J, Timmermans JP, Robberecht W, De Jonghe P, Van Den Bosch L, Janssens S, Timmerman V - Hum. Mol. Genet. (2010)

Mutant HSPB8 induces neurite degeneration in a small proportion of primary sensory neurons. Mouse sensory neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV6 and immunostained at DIV10 using β-III tubulin antibody. Merged confocal micrographs of GFP (green) and β-III tubulin (red) are shown (A–D). No signs of neurite degeneration were found in sensory neurons expressing GFP (A), HSPB8-WT (B) and HSPB8-K141N (C), while the neurites of some cells expressing HSPB8-K141E were clearly beaded (arrow) (D). Scale bar = 10 µm. The incidence of neurite degeneration was quantified by counting the proportion of neurons with beaded neurites (E). **P-value < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2908473&req=5

DDQ234F4: Mutant HSPB8 induces neurite degeneration in a small proportion of primary sensory neurons. Mouse sensory neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV6 and immunostained at DIV10 using β-III tubulin antibody. Merged confocal micrographs of GFP (green) and β-III tubulin (red) are shown (A–D). No signs of neurite degeneration were found in sensory neurons expressing GFP (A), HSPB8-WT (B) and HSPB8-K141N (C), while the neurites of some cells expressing HSPB8-K141E were clearly beaded (arrow) (D). Scale bar = 10 µm. The incidence of neurite degeneration was quantified by counting the proportion of neurons with beaded neurites (E). **P-value < 0.01.
Mentions: The majority of the primary sensory neurons expressing mutant K141N-HSPB8 appeared healthy and morphologically similar to WT-HSPB8 or native GFP expressing sensory neurons (Fig. 4A–C). However, beaded neurites were observed in ∼10% of the mutant K141E-HSPB8 expressing sensory neuronal cells, similarly to what we saw before in motor neurons, though to a lower extent (10 versus 80%) (Fig. 4D) (GFP = 0.0 ± 0.0%, n = 655; WT-HSPB8-GFP = 0.0 ± 0.0%, n = 436; K141N-GFP = 0.5 ± 0.0%, n = 578, P-value = 0.221 and K141E-GFP = 9.7 ± 1.9%, n = 660, P-value = 0.000) (Fig. 4E).Figure 4.

Bottom Line: Furthermore, expression of the K141E (and to a lesser extent, K141N) mutation also induced spheroids in the neurites.While overt in motor neurons, these phenotypes were only very mildly present in sensory neurons and completely absent in cortical neurons.Also glial cells did not show an altered phenotype upon expression of mutant HSPB8.

View Article: PubMed Central - PubMed

Affiliation: Peripheral Neuropathy, VIB Department of Molecular Genetics, University of Antwerp, Antwerp, Belgium.

ABSTRACT
Missense mutations (K141N and K141E) in the alpha-crystallin domain of the small heat shock protein HSPB8 (HSP22) cause distal hereditary motor neuropathy (distal HMN) or Charcot-Marie-Tooth neuropathy type 2L (CMT2L). The mechanism through which mutant HSPB8 leads to a specific motor neuron disease phenotype is currently unknown. To address this question, we compared the effect of mutant HSPB8 in primary neuronal and glial cell cultures. In motor neurons, expression of both HSPB8 K141N and K141E mutations clearly resulted in neurite degeneration, as manifested by a reduction in number of neurites per cell, as well as in a reduction in average length of the neurites. Furthermore, expression of the K141E (and to a lesser extent, K141N) mutation also induced spheroids in the neurites. We did not detect any signs of apoptosis in motor neurons, showing that mutant HSPB8 resulted in neurite degeneration without inducing neuronal death. While overt in motor neurons, these phenotypes were only very mildly present in sensory neurons and completely absent in cortical neurons. Also glial cells did not show an altered phenotype upon expression of mutant HSPB8. These findings show that despite the ubiquitous presence of HSPB8, only motor neurons appear to be affected by the K141N and K141E mutations which explain the predominant motor neuron phenotype in distal HMN and CMT2L.

Show MeSH
Related in: MedlinePlus