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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 abnormalities in primary motor neurons. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at day in vitro 3 (DIV3) and immunostained against the non-phosphorylated epitope in neurofilament H (SMI32 antibody) at DIV7. Non-transduced motor neurons (A), motor neurons expressing native GFP (B) and WT-HSPB8 (C) show a punctate and homogenous neurofilament distribution in the motor neuron soma and normal formation of long and intact neurites, while neuritic processes of motor neurons expressing mutant HSPB8-K141N are shortened (D), and motor neurons expressing mutant HSPB8-K141E exhibited clear signs of neurite degeneration (spheroids or beaded neurites) (E). Scale bar = 10 µm. The incidence of neurite abnormalities in the motor neuron cultures were quantified by counting the proportion of neurons with abnormal (reduced or shortened) neurites (F) and with clear signs of neurite degeneration (beaded neurites) (G), by measuring neurite length distribution (H) and by counting the number of neurites per neuron (I). ns = not significant, **P-value < 0.01.
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DDQ234F1: Mutant HSPB8 induces neurite abnormalities in primary motor neurons. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at day in vitro 3 (DIV3) and immunostained against the non-phosphorylated epitope in neurofilament H (SMI32 antibody) at DIV7. Non-transduced motor neurons (A), motor neurons expressing native GFP (B) and WT-HSPB8 (C) show a punctate and homogenous neurofilament distribution in the motor neuron soma and normal formation of long and intact neurites, while neuritic processes of motor neurons expressing mutant HSPB8-K141N are shortened (D), and motor neurons expressing mutant HSPB8-K141E exhibited clear signs of neurite degeneration (spheroids or beaded neurites) (E). Scale bar = 10 µm. The incidence of neurite abnormalities in the motor neuron cultures were quantified by counting the proportion of neurons with abnormal (reduced or shortened) neurites (F) and with clear signs of neurite degeneration (beaded neurites) (G), by measuring neurite length distribution (H) and by counting the number of neurites per neuron (I). ns = not significant, **P-value < 0.01.

Mentions: Interestingly, striking differences could be observed when we compared the morphology of motor neurons transduced with WT-HSPB8 with those expressing mutant HSPB8, as judged by SMI32 neurofilament immunostaining. Motor neurons transduced with mutant HSPB8 clearly showed neurites with signs of degeneration, while this was not the case in neurons transduced with WT HSPB8 or control constructs (Fig. 1A–E). In cells expressing the K141N-HSPB8-GFP construct, we observed a reduction in length of the distal neurites (defined as shortened neurites in Materials and Methods) in ∼80% of the mutant expressing motor neurons (data obtained at 4 days after transduction and for three independent experiments; percentage motor neurons in culture showing shortened neurites: GFP = 17.0 ± 1.0%, n = 361; WT-HSPB8-GFP = 14.0 ± 2.0%, n = 192; K141N-GFP = 86.0 ± 3.0%, n = 150, P-value = 2.5 × 10−5 and K141E-GFP = 75.0 ± 3.0%, n = 300, P-value = 8.7 × 10−6) (Fig. 1F). In cells expressing the K141E-HSPB8-GFP construct, we observed a more severe phenotype consisting of shortened neurites, as well as neurites containing beaded-like structures resembling spheroids (data of three independent experiments, percentage of motor neurons showing beaded phenotype at day in vitro 7 (DIV7): GFP = 12.0 ± 5.0%, n = 338; WT-HSPB8-GFP = 0.0 ± 0.0%, n = 192; K141N-HSPB8-GFP = 9.0 ± 6.0%, n = 150, P-value = 0.1333 and K141E-HSPB8-GFP = 90.0 ± 1.0%, n = 300, P-value = 4.1 × 10−5) (Fig. 1G).Figure 1.


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 abnormalities in primary motor neurons. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at day in vitro 3 (DIV3) and immunostained against the non-phosphorylated epitope in neurofilament H (SMI32 antibody) at DIV7. Non-transduced motor neurons (A), motor neurons expressing native GFP (B) and WT-HSPB8 (C) show a punctate and homogenous neurofilament distribution in the motor neuron soma and normal formation of long and intact neurites, while neuritic processes of motor neurons expressing mutant HSPB8-K141N are shortened (D), and motor neurons expressing mutant HSPB8-K141E exhibited clear signs of neurite degeneration (spheroids or beaded neurites) (E). Scale bar = 10 µm. The incidence of neurite abnormalities in the motor neuron cultures were quantified by counting the proportion of neurons with abnormal (reduced or shortened) neurites (F) and with clear signs of neurite degeneration (beaded neurites) (G), by measuring neurite length distribution (H) and by counting the number of neurites per neuron (I). ns = not significant, **P-value < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2908473&req=5

DDQ234F1: Mutant HSPB8 induces neurite abnormalities in primary motor neurons. Rat motor neurons were transduced with pLenti-GFP, pLenti-WT-HSPB8-GFP or mutant pLenti-K141N/K141E-HSPB8-GFP constructs at day in vitro 3 (DIV3) and immunostained against the non-phosphorylated epitope in neurofilament H (SMI32 antibody) at DIV7. Non-transduced motor neurons (A), motor neurons expressing native GFP (B) and WT-HSPB8 (C) show a punctate and homogenous neurofilament distribution in the motor neuron soma and normal formation of long and intact neurites, while neuritic processes of motor neurons expressing mutant HSPB8-K141N are shortened (D), and motor neurons expressing mutant HSPB8-K141E exhibited clear signs of neurite degeneration (spheroids or beaded neurites) (E). Scale bar = 10 µm. The incidence of neurite abnormalities in the motor neuron cultures were quantified by counting the proportion of neurons with abnormal (reduced or shortened) neurites (F) and with clear signs of neurite degeneration (beaded neurites) (G), by measuring neurite length distribution (H) and by counting the number of neurites per neuron (I). ns = not significant, **P-value < 0.01.
Mentions: Interestingly, striking differences could be observed when we compared the morphology of motor neurons transduced with WT-HSPB8 with those expressing mutant HSPB8, as judged by SMI32 neurofilament immunostaining. Motor neurons transduced with mutant HSPB8 clearly showed neurites with signs of degeneration, while this was not the case in neurons transduced with WT HSPB8 or control constructs (Fig. 1A–E). In cells expressing the K141N-HSPB8-GFP construct, we observed a reduction in length of the distal neurites (defined as shortened neurites in Materials and Methods) in ∼80% of the mutant expressing motor neurons (data obtained at 4 days after transduction and for three independent experiments; percentage motor neurons in culture showing shortened neurites: GFP = 17.0 ± 1.0%, n = 361; WT-HSPB8-GFP = 14.0 ± 2.0%, n = 192; K141N-GFP = 86.0 ± 3.0%, n = 150, P-value = 2.5 × 10−5 and K141E-GFP = 75.0 ± 3.0%, n = 300, P-value = 8.7 × 10−6) (Fig. 1F). In cells expressing the K141E-HSPB8-GFP construct, we observed a more severe phenotype consisting of shortened neurites, as well as neurites containing beaded-like structures resembling spheroids (data of three independent experiments, percentage of motor neurons showing beaded phenotype at day in vitro 7 (DIV7): GFP = 12.0 ± 5.0%, n = 338; WT-HSPB8-GFP = 0.0 ± 0.0%, n = 192; K141N-HSPB8-GFP = 9.0 ± 6.0%, n = 150, P-value = 0.1333 and K141E-HSPB8-GFP = 90.0 ± 1.0%, n = 300, P-value = 4.1 × 10−5) (Fig. 1G).Figure 1.

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