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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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Motor neurons expressing mutant HSPB8 show enhanced neuritic APP accumulation. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV3 and immunostained using amyloid precursor protein (APP) antibody at DIV7. Motor neuron neurites expressing GFP (A) or HSPB8-WT (B) mainly show a faint and punctate appearance of APP in the neurites, similar to non-transduced neurites, while neurites of motor neuron expressing HSPB8-K141N (C) or HSPB8-K141E (D) accumulate more APP (arrow). The incidence of APP accumulation was quantified by counting the proportion of cells with strong neuritic APP accumulation (E). **P-value < 0.01. Scale bar = 10 µm.
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DDQ234F2: Motor neurons expressing mutant HSPB8 show enhanced neuritic APP accumulation. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV3 and immunostained using amyloid precursor protein (APP) antibody at DIV7. Motor neuron neurites expressing GFP (A) or HSPB8-WT (B) mainly show a faint and punctate appearance of APP in the neurites, similar to non-transduced neurites, while neurites of motor neuron expressing HSPB8-K141N (C) or HSPB8-K141E (D) accumulate more APP (arrow). The incidence of APP accumulation was quantified by counting the proportion of cells with strong neuritic APP accumulation (E). **P-value < 0.01. Scale bar = 10 µm.

Mentions: To further verify if the neurite degeneration phenotype that we observed in motor neuron could be a sign of axonal degeneration, we used amyloid precursor protein (APP) as a marker for neurodegeneration. Early studies identified APP as a kinesin-binding protein (12,13). APP is a normal component of neurons transported by fast axonal transport, and specifically accumulates to detectable levels at sites where the cytoskeleton breaks down and therefore can be used as a read-out to study axonal transport defects (14–16). In healthy neuronal cells with normal axonal transport rates, APP is rapidly transported, leading to disperse signals on APP immunostainings. In cells with axonal transport defects, APP rapidly accumulates leading to a strong signal in immunostainings. It is well established that defects in axonal transport and neurite degeneration are strongly linked (17) and therefore we examined this by immunostaining APP in neurites of motor neurons expressing either WT or mutant HSPB8 (Fig. 2A and B). Motor neurons expressing HSPB8-WT showed mostly a faint and punctate pattern of APP immunostaining indicative of a normal axonal transport, while strong accumulation of APP was detected in many neurites of mutant HSPB8 expressing motor neurons (data of three independent experiments at DIV7: GFP = 12 ± 1.0%, n = 994; WT-HSPB8-GFP = 12 ± 0.0%, n = 971; K141N-GFP = 24.5 ± 2.0%, n = 864, P-value = 0.0082 and K141E-GFP = 25 ± 1.0%, n = 1029, P-value = 0.0019) (Fig. 2C–E). These data therefore suggest that axonal transport blockage could be implicated in the development of mutant HSPB8 associated neurite degeneration.Figure 2.


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)

Motor neurons expressing mutant HSPB8 show enhanced neuritic APP accumulation. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV3 and immunostained using amyloid precursor protein (APP) antibody at DIV7. Motor neuron neurites expressing GFP (A) or HSPB8-WT (B) mainly show a faint and punctate appearance of APP in the neurites, similar to non-transduced neurites, while neurites of motor neuron expressing HSPB8-K141N (C) or HSPB8-K141E (D) accumulate more APP (arrow). The incidence of APP accumulation was quantified by counting the proportion of cells with strong neuritic APP accumulation (E). **P-value < 0.01. Scale bar = 10 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDQ234F2: Motor neurons expressing mutant HSPB8 show enhanced neuritic APP accumulation. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at DIV3 and immunostained using amyloid precursor protein (APP) antibody at DIV7. Motor neuron neurites expressing GFP (A) or HSPB8-WT (B) mainly show a faint and punctate appearance of APP in the neurites, similar to non-transduced neurites, while neurites of motor neuron expressing HSPB8-K141N (C) or HSPB8-K141E (D) accumulate more APP (arrow). The incidence of APP accumulation was quantified by counting the proportion of cells with strong neuritic APP accumulation (E). **P-value < 0.01. Scale bar = 10 µm.
Mentions: To further verify if the neurite degeneration phenotype that we observed in motor neuron could be a sign of axonal degeneration, we used amyloid precursor protein (APP) as a marker for neurodegeneration. Early studies identified APP as a kinesin-binding protein (12,13). APP is a normal component of neurons transported by fast axonal transport, and specifically accumulates to detectable levels at sites where the cytoskeleton breaks down and therefore can be used as a read-out to study axonal transport defects (14–16). In healthy neuronal cells with normal axonal transport rates, APP is rapidly transported, leading to disperse signals on APP immunostainings. In cells with axonal transport defects, APP rapidly accumulates leading to a strong signal in immunostainings. It is well established that defects in axonal transport and neurite degeneration are strongly linked (17) and therefore we examined this by immunostaining APP in neurites of motor neurons expressing either WT or mutant HSPB8 (Fig. 2A and B). Motor neurons expressing HSPB8-WT showed mostly a faint and punctate pattern of APP immunostaining indicative of a normal axonal transport, while strong accumulation of APP was detected in many neurites of mutant HSPB8 expressing motor neurons (data of three independent experiments at DIV7: GFP = 12 ± 1.0%, n = 994; WT-HSPB8-GFP = 12 ± 0.0%, n = 971; K141N-GFP = 24.5 ± 2.0%, n = 864, P-value = 0.0082 and K141E-GFP = 25 ± 1.0%, n = 1029, P-value = 0.0019) (Fig. 2C–E). These data therefore suggest that axonal transport blockage could be implicated in the development of mutant HSPB8 associated neurite degeneration.Figure 2.

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