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Human Molecular Genetics Advance Access originally published online on December 20, 2005
Human Molecular Genetics 2006 15(2):347-354; doi:10.1093/hmg/ddi452
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A mutation in the small heat-shock protein HSPB1 leading to distal hereditary motor neuronopathy disrupts neurofilament assembly and the axonal transport of specific cellular cargoes

Steven Ackerley, Paul A. James, Arran Kalli, Sarah French, Kay E. Davies and Kevin Talbot*

Department of Human Anatomy and Genetics, South Parks Road, Oxford OX1 3QX, UK

* To whom correspondence should be addressed. Tel: +44 1865282827; Fax: +44 1865272420; Email: kevin.talbot{at}clneuro.ox.ac.uk

Received September 5, 2005; Accepted December 9, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Distal hereditary motor neuronopathies (dHMNs) are a clinically and genetically heterogeneous group of disorders in which motor neurons selectively undergo age-dependant degeneration. Mutations in the small heat-shock protein HSPB1 (HSP27) are responsible for one form of dHMN. In this study, we have analysed the effect of expressing a form of mutant HSPB1 in primary neuronal cells in culture. Mutant (P182L) but not wild-type HSPB1 led to the formation of insoluble intracellular aggregates and to the sequestration in the cytoplasm of selective cellular components, including neurofilament middle chain subunit (NF-M) and p150 dynactin. These findings suggest a possible pathogenic mechanism for HSPB1 whereby the mutation may lead to preferential motor neuron loss by disrupting selective components essential for axonal structure and transport.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The distal hereditary motor neuronopathies (dHMNs) are a group of genetically determined disorders characterized by weakness and wasting, particularly of the distal muscles of both upper and lower limbs and slow, steady progression leading eventually to difficulty in ambulation and fine motor tasks. Clinically and electrophysiologically, affected patients appear to have pure lower motor neuron degeneration such as that seen in various forms of spinal muscular atrophy (SMA). Some authors consider dHMN to be a pure motor, or ‘spinal’, form of the Charcot–Marie–Tooth (CMT)-inherited neuropathies, as the pattern of motor involvement is similar to that seen in CMT (1Go). Recently, the clinical and genetic heterogeneity of pure lower motor neuron degeneration and the overlap with axonal forms of CMT have been confirmed by the identification of a number of genes in which mutation leads to dHMN/SMA (2Go). Missense mutations in the gene encoding the 27 kDa small heat-shock protein B1 (HSPB1, previously known as HSP27) were identified in a number of families with either dHMN or axonal CMT (3Go).

The heat-shock proteins are a large family of molecular chaperones, which can be further subdivided into groups including that of the small heat-shock proteins (sHSP) (4Go). Members of the sHSP family, which includes HSPB1, are defined by a low molecular weight and the presence of an approximately 80–100 amino acid region known as the {alpha}-crystallin domain (5Go). A number of roles have been identified for HSPB1, which include both anti-apoptotic functions and interactions with various components of the cytoskeleton (6Go–8Go). A number of heat-shock proteins including HSPB1 have been demonstrated to be upregulated in various neurodegenerative diseases, and interestingly, HSPB1 has also been shown to be essential for the survival of both sensory and motor neurons (4Go,9Go).

A number of mutations have been identified in HSPB1 with all but one being present within the conserved {alpha}-crystallin domain, a region which is known to be involved in the formation of higher order structures (3Go). Disease-causing mutations have also been identified within the {alpha}-crystallin domain of other sHSP including HSPB8 (also known as HSP22) and {alpha}B-crystallin, which result in dHMN and desmin-related myopathy, respectively (10Go,11Go). Outside of the {alpha}-crystallin domain, a further mutation resulting in a proline to leucine substitution at amino acid 182 (P182L) has been identified within the C-terminal tail of HSPB1, the region possessing the greatest variability across all sHSP family members (5Go). The pathogenic mechanism of this mutation is not known. Here we demonstrate that, unlike the wt protein, the P182L mutant HSPB1 forms large aggregates within cell bodies and selectively disrupts transport processes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutant HSPB1 forms aggregates and fails to undergo transport down neuronal processes
We have analysed the effect of the recently identified HSPB1 P182L mutation on the subcellular distribution of the protein compared to wild-type (wt) HSPB1 in transfected primary cortical cultures. HA-tagged wt HSPB1 was found to be present in a general diffuse, finely granular pattern in both the cell body and neurites in 93% of transfected cortical cells examined. However, the HA-tagged P182L mutant HSPB1 was found to have a markedly different appearance and form aggregates, predominately in the cell body, with little mutant protein appearing in neurites in 92% of cells examined (Fig. 1A and B). A small number of wt HSPB1 expressing cells appeared to contain aggregates in the cell body (7%) possibly due to the overexpression of the protein. However, these aggregates appeared fewer in number and smaller in size than those produced by P182L HSPB1, and even in such wt HSPB1 aggregate-containing cells as were observed, the overall distribution of HA-tagged wt HSPB1 appeared unchanged and was still present throughout the length of neurites (data not shown). All transfected cells appeared healthy and there were no signs of cell death in either wt or mutant (P182) HSPB1-transfected cells as judged by general cellular morphology and visualization of nuclei stained with DAPI. A similar cellular distribution for HA-tagged wt or P182L HSPB1 was also found in both transfected HEK293 and N2a cell lines (data not shown).



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  		Figure 1. Expression of mutant but not wt HSPB1 results in the formation of insoluble aggregates in transfected cells. Primary cortical cultures were transfected with either wt (A) or mutant P182L (B) HA-tagged HSPB1 and stained with an antibody against the HA tag. Scale bar=20 µm. (C) Triton X-100 soluble (Sol) and insoluble (Insol) fractions were prepared from N2a cells transfected with either wt or mutant P182L HA-tagged HSPB1, NT are non-transfected cells.

 
Formation of detergent insoluble aggregates has been reported as an aberrant property of a number of proteins found to be mutated in neurodegenerative diseases including superoxide dismutase 1 (SOD1) (12Go,13Go). We therefore performed Triton X-100 soluble/insoluble preparations of N2a cells transfected with either HA-tagged wt or P182L mutant HSPB1. The majority of transfected HA-tagged wt HSPB1 was found to be present in the Triton X-100 soluble fraction with only a small amount present in the insoluble fraction, whereas the mutant P182L HSPB1 was found exclusively in the Triton X-100 insoluble fraction (Fig. 1C).

Expression of green fluorescent protein (GFP) has been used in many studies as a means to outline the shape and processes of individual transfected cells (14Go). We therefore co-transfected either HA-tagged wt or mutant P182L HSPB1 with a plasmid expressing GFP (pEGFP-N1) to assess the extent to which wt or mutant HSPB1 had been transported within the neurites of cortical cultures. For analysis of the distance travelled by HA-tagged wt or mutant P182L HSPB1, all images of transfected cells were captured at the same exposure and the distance travelled was defined as the furthest point at which fluorescence above background was detected in any given transfected cell. Following transfection (16 h), the wt HSPB1 was transported within neurites and was uniformly present down the entire length of cell processes as outlined by GFP expression (Fig. 2A and B). However, the P182L mutant HSPB1 failed to be transported to the same extent and was present only in the cell body and proximal portion of neurites (Fig. 2C and D). Analysis of the distance travelled revealed that HA-tagged wt HSPB1 had travelled significantly further than the HA-tagged mutant P182L HSPB1 (Fig. 2E).



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  		Figure 2. Mutant but not wt HSPB1 fails to be transported within the neurites of primary cortical cultures. Cells were co-transfected with pEGFP-N1 and either wt (A and B) or mutant P182L (C and D) HA-tagged HSPB1. Transfected HA-tagged HSPB1 (A and C) is detected using antibody against the HA tag, whereas (B) and (D) show GFP fluorescence. Scale bar=40 µm. (E) Distance travelled 16 h post-transfection by either wt or mutant P182L HA-tagged HSPB1 in cells co-transfected with pEGFP-N1. Student's t-test revealed significant difference in the distance travelled by mutant P182L HSPB1 (P<0.01).

 
Wt HSPB1 fails to prevent aggregation of mutant HSPB1 and is sequestered to cell bodies in co-transfected cells
HSPB1 has been shown to be capable of preventing the formation of aggregates resulting from mutation in the sHSP {alpha}B-crystallin (15Go). We therefore co-transfected either wt or mutant P182L HA-tagged HSPB1 with a GFP-tagged wt HSPB1 (GFP–HSPB1) construct to assess any effect overexpression of wt HSPB1 may have on P182L aggregate formation. Both HA-tagged and GFP-tagged wt HSPB1 had a similar overlapping distribution in co-transfected cells (Fig. 3A and B). The majority of cells appeared similar to that of cells transfected with only HA-tagged HSPB1 with both the GFP-tagged and HA-tagged wt HSPB1 proteins having a diffuse, finely granular appearance and even distribution down the length of neurites. However, in cells transfected with mutant P182L HSPB1, the distribution of co-transfected GFP–HSPB1 was markedly disrupted and was found to strongly co-localize with the cell body aggregates of mutant (P182L) HSPB1 (Fig. 3C and D). There was, however, no significant reduction in the formation of aggregates by mutant P182L HSPB1 in cells co-transfected with GFP–HSPB1 with 92% of cells still containing aggregates (Fig. 3E).



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  		Figure 3. Expression of wt HSPB1 fails to prevent aggregation of mutant P182L HSPB1. Cells were co-transfected with GFP-tagged wt HSPB1 and either wt (A and B) or mutant P182L (C and D) HA-tagged HSPB1. Transfected HA-tagged HSPB1 (A and C) is detected using antibody against the HA tag, whereas (B) and (D) show GFP-tagged wt HSPB1. Scale bar=20 µm. (E) Quantification of numbers of co-transfected cells containing aggregates. (F) Distance travelled by GFP-tagged wt HSPB1 in cells co-transfected with either HA-tagged wt or mutant P182L HSPB1. The distance travelled by HA-tagged wt and GFP-tagged wt HSPB1 or HA-tagged mutant P182L HSPB1 and GFP-tagged wt HSPB1 was measured in co-transfected cells. Student's t-test revealed significant difference in the distance travelled by GFP-tagged wt HSPB1 due to expression of mutant P182L HSPB1 (P<0.01).

 
This sequestration to cell body aggregates also appeared to result in a marked reduction of GFP–HSPB1 in neurites of cells co-transfected with mutant P182L HSPB1. Indeed the distance travelled by GFP–HSPB1 in cells co-transfected with mutant P182L HA-tagged HSPB1 was significantly less than that travelled by GFP–HSPB1 in cells co-transfected with wt HA-tagged HSPB1 (Fig. 3F). As with overexpression of HA-tagged HSPB1 (discussed above), there were aggregates in some cells co-transfected with GFP-tagged and HA-tagged wt HSPB1 (12%); however, as noted above, these aggregates were fewer in number and smaller in size, and even in such aggregate-containing cells, the overall distribution of either GFP-tagged or HA-tagged wt HSPB1 appeared unchanged and was still present throughout the length of neurites (data not shown). These data suggest that mutant HSPB1 sequestrates wt HSPB1 and prevents its transport down neuronal processes.

Mutant HSPB1 disrupts the filament formation of GFP-NF-M
As part of the family of sHSP, HSPB1 is known to have chaperone-like activity and has also been demonstrated to interact with various components of the cytoskeleton including members of the intermediate filament protein family (8Go). Recently, another CMT/dHMN-related HSPB1 mutant (S135F) has been demonstrated to disrupt the assembly of neurofilaments (NFs) in a cell line (3Go). We have further investigated this process in primary cortical cultures using a GFP-tagged neurofilament middle chain (GFP-NF-M) fusion protein that has previously been used to investigate NF transport and has been demonstrated to assemble into the endogenous NF network (14Go).

Co-transfection of HA-tagged wt HSPB1 and GFP-NF-M into cortical cells resulted in GFP-NF-M which could be visualized as a fine filamentous network in 90% of cells that were co-transfected (Fig. 4A and B). The co-transfected GFP-NF-M was also present down the length of neurites. However, similar to previous studies, we found that the co-expression of HA-tagged mutant P182L HSPB1 with GFP-NF-M appeared to result in the disruption of GFP-NF-M assembly and incorporation into the endogenous NF network in the majority of cells containing mutant P182L HSPB1 aggregates (92%) (Fig. 4C and D). Rather than appearing as a fine filamentous network, GFP-NF-M was present in an amorphous, non-filamentous pattern in cell bodies, which in some cells contained aggregates that co-localized with mutant P182L HSPB1 either in the cell bodies or in the proximal portion of neurites. The GFP-NF-M in cells co-transfected with mutant P182L HSPB1 also failed to extend into neurites in many of the cells. In the small number of cells that contained aggregates of HA-tagged wt HSPB1 (%), there appeared to be no disruption or co-localization with the transfected GFP-NF-M (data not shown).



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  		Figure 4. Expression of mutant but not wt HSPB1 results in the disruption of NF assembly in transfected cells. Cells were co-transfected with GFP-tagged NF-M and either wt (A and B) or mutant P182L (C and D) HA-tagged HSPB1. Transfected HA-tagged HSPB1 (A and C) is detected using antibody against the HA tag, whereas (B) and (D) show GFP-tagged NF-M expression. Scale bar=20 µm.

 
Mutant HSPB1 selectively disrupts the transport of specific cellular cargoes
Abnormal protein aggregation and disruption of axonal transport have become favoured potential mechanistic processes in a number of neurodegenerative diseases including CMT disease and motor neuron diseases (16Go). We therefore decided to investigate whether the aberrant accumulation of mutant P182L HSPB1 in cell bodies might perturb the transport of cellular components known to be either involved in axonal transport or present within axons and/or synapses. The dynein/dynactin complex is involved in retrograde axonal transport, and disruption to components of this complex, including mutation of the p150 subunit of dynactin, has been demonstrated to result in motor neuron disease (17Go–19Go). Co-transfection of a GFP-tagged p150 fusion protein (GFP–p150) with HA-tagged wt HSPB1 produced a pattern of GFP–p150 distribution which was unchanged from that seen following the expression of GFP–p150 only. The GFP–p150 was found to be distributed both in the cell body and also along the length of neurites and presumably due to association with microtubules had a slightly filamentous appearance (Fig. 5A and B). Abnormal aggregation of GFP–p150 following transfection with HA-tagged wt HSPB1 was only seen in a small number of cells (3%) and never appeared to co-localize with wt HSPB1. In contrast, the co-expression of GFP–p150 with HA-tagged mutant P182L HSPB1 resulted in altered localization of GFP–p150 with aggregation of GFP–p150 within cell bodies (96%) (Fig. 5C and D). These aggregates of GFP–p150 co-localized with the aggregated mutant P182 HSPB1 in cell bodies. The appearance of GFP–p150 within axons of cells co-transfected with mutant P182L HSPB1 was typical at low levels.



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  		Figure 5. Expression of mutant but not wt HSPB1 results in the disruption of p150 distribution in transfected cells. Cells were co-transfected with GFP-tagged p150 and either wt (A and B) or mutant P182L (C and D) HA-tagged HSPB1. Transfected HA-tagged HSPB1 (A and C) is detected using antibody against the HA tag, whereas (B) and (D) show GFP-tagged p150 expression. Scale bar=20 µm. (E) Triton X-100 soluble (Sol) and insoluble (Insol) fractions were prepared from N2a cells transfected with GFP–p150 and either wt or mutant P182L HA-tagged HSPB1 and probed with an antibody against p150. NT are non-transfected cells, MB is mouse brain.

 
We decided to further investigate biochemically the extent to which p150 distribution may be altered due to mutant P182L HSPB1 aggregates by probing Triton X-100 soluble/insoluble fractions from N2a cells transfected with GFP–p150 and either wt or mutant P182L HA-tagged HSPB1 with an antibody directed against p150. As with non-transfected cells the p150 was found in the Triton X-100 soluble fraction in cells co-transfected with GFP–p150 and HA-tagged wt HSPB1, with only a small amount present in the insoluble fraction (Fig. 5E). However, although p150 was still present in the soluble fraction from cells co-transfected with GFP–p150 and P182L HA-tagged HSPB1, a greater proportion was present in the Triton X-100 insoluble fraction than was found in the insoluble fraction from either non-transfected or wt HA-tagged HSPB1 transfected cells (Fig. 5E).

In order to investigate whether this was a specific effect of mutant P182L HSPB1 on a component of the dynein/dynactin complex or a more general effect on axonal transport, we decided to look at the effect of mutant P182L HSPB1 on the transport of mitochondria. The transport of mitochondria has previously been successfully used to investigate disruption of axonal transport in a cell culture model (20Go). However, following transfection of cortical cultures with the mitochondrial marker DsRed2-Mito and either wt or mutant P182L HA-tagged HSPB1, we found no significant difference in the number of mitochondria present within a defined region between the two groups (Fig. 6A–E), with the number of mitochondria present within this segment comparable with previous studies (20Go). Additionally, unlike GFP-NF-M or GFP–p150, there was no evidence of mitochondria co-localizing with or being trapped within mutant P182L HSPB1 containing aggregates.



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  		Figure 6. Mutant HSPB1 has no effect on the anterograde transport of mitochondria within the neurites of primary cortical cultures. Cells were co-transfected with DsRed2-Mito (DS-Mito) and either wt (A and B) or mutant P182L (C and D) HA-tagged HSPB1. Transfected HA-tagged HSPB1 (A and C) is detected using antibody against the HA tag, whereas (B) and (D) show DsRed2-Mito fluorescence. Scale bar=20 µm. (E) Quantification of mitochondria distribution in a segment of axon 100 µm in length beginning 50 µm from the cell body. Student's t-test revealed no significant difference in the distribution of mitochondria between wt HSPB1 and mutant P182L HSPB1 expressing neurons (P>0.5).

 
We further investigated the effect of mutant P182L HSPB1 on axonal transport using synaptotagmin I, a vesicle-associated protein which is known to be actively transported within axons (21Go). Co-transfection of a GFP-tagged synaptotagmin I fusion protein (GFP–SYT I) with HA-tagged wt HSPB1 resulted in a general diffuse GFP–SYT I expression pattern with GFP–SYT I present in both the cell body and neurites with a distribution which did not differ from that of GFP–SYT I only transfected cells (Fig. 7A and B) (data not shown). Interestingly, co-transfection of GFP–SYT I with HA-tagged mutant P182L HSPB1 also did not produce a pattern of GFP–SYT I distribution that looked different from GFP–SYT I only transfected cells (Fig. 7C and D). Although the mutant P182L HSPB1 was still present as aggregates within the cell body and proximal region of processes there was no co-localization with GFP–SYT I.



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  		Figure 7. Expression of mutant HSPB1 has no effect on synaptotagmin distribution in transfected cells. Cells were co-transfected with GFP-tagged synaptotagmin and either wt (A and B) or mutant P182L (C and D) HA-tagged HSPB1. Transfected HA-tagged HSPB1 (A and C) is detected using antibody against the HA tag, whereas (B) and (D) show GFP-tagged synaptotagmin expression. Scale bar=20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Protein misfolding or abnormal aggregation has been identified as a characteristic feature in a number of neurodegenerative diseases including motor neuron diseases (4Go,5Go). The large family of heat-shock proteins, which includes the subgroup of sHSP, are molecular chaperones that have been demonstrated to be upregulated in a number of neurodegenerative diseases as a mechanism to combat the abnormal protein aggregation or misfolding found in the disease state (4Go). Recently, disease-causing mutations within HSPB1, a member of the family of sHSP, were identified in a number of families diagnosed as having either dHMN or CMT disease (3Go). The pathogenic mechanism of the mutations is however not yet understood. Here, we have shown in primary cortical neurons that the recently identified C-terminal mutation (P182L) in HSPB1 results in the abnormal aggregation of the mutant protein when compared with wt HSPB1. This is a similar result to that demonstrated for two other sHSP, HSP22 and {alpha}B-crystallin for which identified mutations have also been shown to result in the abnormal aggregation of mutant protein (10Go,11Go).

Overexpression of wt HSPB1 has previously been demonstrated to be sufficient to result in the solubilization of aggregates resulting from a mutation in the sHSP {alpha}B-crystallin (15Go). Here we have found that co-transfection of wt HSPB1 with mutant P182L HSPB1 results in the strong co-localization of wt HSPB1 into the intracellular aggregates present within cell bodies with no change in the amount of cells displaying aggregates. This sequestering of wt HSPB1 into intracellular aggregates could remove a portion of wt HSPB1 from other essential functions within the cell. This could be of particular significance in specific cell types where a basal level of HSPB1 expression may be critical, such as sensory or motor neurons where HSPB1 has previously been demonstrated to be constitutively expressed and essential for cell survival (9Go,22Go). Indeed, one recently proposed toxic mechanism for mutant SOD1 involves the aberrant binding of mutant but not of wt SOD1 to HSPB1 thus depleting the amount of HSPB1 available to perform essential cellular functions. It has also been shown that HSPB1 is actively transported within neurons and is present in the axon and growth cone (23Go–25Go). Although the function of HSPB1 within axons is still not clear, it is interesting that mutant HSPB1 fails to be fully transported into neurites of transfected cells, which again could possibly exclude HSPB1 from an essential function in either processes or termini.

A number of roles have been identified for the sHSP including regulation and maintenance of various components of the cytoskeleton (8Go,26Go). HSPB1 has been shown to interact with various members of the intermediate filament family, and recently another CMT disease related mutation in HSPB1 was shown to result in disruption of the NF assembly (3Go). Here again, we have demonstrated that expression of a mutant HSPB1 results in disruption of the NF network in cortical neurons, whereas overexpression of wt HSPB1 does not have the same effect. A similar result is also seen for {alpha}B-crystallin where mutations result in disruption and aggregation of desmin intermediate filaments, leading to desmin-related myopathy (11Go,27Go). A number of studies have demonstrated that larger neuronal cell types, such as motor neurons are particularly vulnerable to altered NF states, and disordered NF assembly resulting in NF aggregates are a pathological hallmark of amyotrophic lateral sclerosis (ALS) (28Go–30Go). Additionally, mutations in the NF light chain which result in disordered NF assembly and aggregation are responsible for disease in some CMT-affected families (31Go,32Go). Therefore, the possibility that the disruption of NF assembly or NF aggregation has a role in the selective pathogenic mechanism of mutant HSPB1 is particularly appealing.

Defects in axonal transport have also been reported as a mechanism in various neurodegenerative diseases (16Go). A number of disease-causing mutations have been identified in components of the axonal transport machinery in both CMT and motor neuron diseases, including mutations in components of the dynein/dynactin complex such as p150 (17Go). The function of p150 in neuronal cells includes providing a link between many cellular cargoes and the retrograde molecular motor dynein and also regulating the processivity of dynein once in motion (33Go). We have shown here that mutant but not wt HSPB1 co-localizes with p150 resulting in the mislocalization of p150 within cell bodies. These mutant HSPB1 containing aggregates appear to sequester p150 and our biochemical data demonstrate that a greater proportion of p150 becomes trapped in the detergent insoluble fraction due to the expression of mutant HSPB1 when compared with cells expressing wt HSPB1. An increased insolubility of various components involved in axonal transport has previously been reported in a Drosophila model system where expression of a mutant-expanded polyQ huntingtin protein results in disruption of axonal transport (34Go). One consequence of this increase in the amount of insoluble p150 could be a reduction in the amount of p150 available within axons or synaptic termini. The retrograde transport of many molecules has been shown to be essential for cellular functioning and survival, and the effect of sequestering p150 in cell bodies could over a period of time result in a decrease in the amount of essential cargoes, such as growth factors or signalling molecules that are translocated back to the cell body. Disruption to such processes may leave cells which place a high demand on axonal transport due to abnormally long axons, such as motor neurons, particularly susceptible. Indeed, a previously identified mutation in p150 dynactin, which results in a motor neuron disease has been shown to disrupt the normal localization of p150 resulting in cell body aggregates similar to those we have observed due to expression of mutant HSPB1 (18Go). In addition, mutations in SOD1, a defined cause of ALS, result in a defect in retrograde transport, and aggregates of mutant SOD1 have been shown to contain the retrograde molecular motor dynein (35Go).

Mitochondria have previously been shown to be actively transported within neurites, and although mitochondrial distribution has been shown to be disrupted in other culture models as a consequence of expressing disease-causing genes, we did not observe a defect in the presence of mutant HSPB1 (20Go). The lack of any effect of mutant HSPB1 on the transport of mitochondria could be due either to mutant HSPB1 having no effects on anterograde axonal transport or perhaps only specific anterograde cargoes being disrupted. We also found no effect on the distribution and transport of synaptotagmin I, another protein known to be anterogradely transported, due to expression of mutant HSPB1. Therefore, rather than the aggregates of mutant HSPB1 non-specifically disrupting axonal transport, they may selectively disrupt particular components essential to particular cells.

In summary, we have shown that mutant but not wt HSPB1 fails to be transported within neurites of cortical neurons and rather forms intracellular aggregates. In addition, we have demonstrated that NF assembly and specific aspects of axonal transport are affected by mutant HSPB1 but not by wt HSPB1 suggesting possible pathogenic mechanisms for this mutation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
All of the experiments described in Results were performed a minimum of three times with similar results.

Plasmids
An HA tag was added to the N-terminus of the human cDNA encoding full-length HSPB1 by PCR. The N-terminal HA-tagged human HSPB1 cDNA was then used to generate mutant P182L HSPB1 using the primer sets 5'-CCATCCTAGTCACCTTCGAG-3' and 5'-ACTCGAGTTACTTGGCGGCAGTCTCATC-3' and 5'-CGAAGGTGACTAGGATGGTG-3' and 5'-AGTCGACGGCAGCATGTACCCATACGACGTACCAG-3', following which the two PCR products were used together as template with the primers 5'-AGTCGACGGCAGCATGTACCCATACGACGTACCAG-3' and 5'-ACTCGAGTTACTTGGCGGCAGTCTCATC-3' to generate the full-length N-terminal HA-tagged P182L HSPB1. Both wt and mutant cDNA were then cloned into pCAGGS for expression studies. Gfp-tagged NF-M, p150 and wt HSPB1 were as previously described (3Go,14Go,36Go). Mitochondria were visualized using pDsRed2-Mito (Clontech) and the shape of neurons using pEGFP-N1 (Clontech).

Primary cell culture, transfection and immunofluorescence microscopy
Primary cortical neurons were obtained from E16 mouse embryos and cultured on glass cover slips coated with poly-D-lysine in 12-well plates. Cells were grown in Neurobasal medium and B27 supplement (Invitrogen) containing 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine. Cells were transfected at 4 DIV using lipofectamine 2000 (Invitrogen). Briefly, half the volume of Neurobasal/B27 medium was removed from cells following which 75 µl of Optimem (invitrogen) containing 250 ng plasmid DNA and 1 µl Lipofectamine 2000 (Invitrogen) was incubated with the cells for 2 h. The media were then gently removed from the cells and replaced with the conditioned Neurobasal/B27 medium.

Cells were fixed and processed for immunofluorescence microscopy either 20 or 44 h post-transfection. Briefly, cells were gently washed once in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized in 0.1% (v/v) Triton X-100 in PBS for 10 min, blocked with 5% (v/v) FBS in PBS for 1 h and then probed using antibody rabbit anti-HA (Sigma) diluted in blocking solution which is directed against the HA tag. Primary antibody was then detected using goat anti-rabbit immunoglobulins (Igs) coupled to Alexa Fluor 594 (Molecular probes) with all other transfected proteins visualized by their GFP tag. The samples were mounted in Vectashield containing DAPI (Vector Labs). Cells were examined using a fluorescent microscope (Leica) and images collected via an AxioCam HRc camera (Zeiss) and processed using AxioVision software (Zeiss).

The presence or absence of aggregates in transfected cells with scored using immunocytochemistry in no fewer than 200 randomly selected cells for each condition. The data presented represent the mean value for three independent experiments. For the analysis of distance travelled, not fewer than 50 cells were assessed for each experimental condition and each experiment was performed at least three times. The data presented are from a representative experiment.

Triton X-100 soluble/insoluble preparations
N2a cells were grown in Dulbecco's modified Eagle's medium (Sigma) containing 5% (v/v) FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine and transfected using lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. For preparation of Triton X-100-soluble and -insoluble fractions, cells were scraped into ice-cold Tris-buffer (pH 7.6) containing 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100 and complete protease inhibitor cocktail (Sigma). Samples were then left on ice for 45 min following which they were spun at 10 000g for 10 min at 4°C. Supernatants and pellets were separated and prepared for SDS–PAGE by addition of SDS–PAGE sample buffer and heating at 100°C. Equal amounts of either Triton X-100-soluble or -insoluble fractions were loaded and analysed by SDS–PAGE and immunoblotting. HA-tagged HSPB1 or p150 was detected using rat anti-HA (Roche) or mouse anti-p150glued (Transduction Laboratories) and visualized following incubation in the appropriate HRP-conjugated anti-rat (Jackson ImmunoResearch Laboratories) or anti-mouse (Amersham) Igs using an enhanced chemiluminescence system (Amersham).

Quantification of mitochondria distribution
For analyses of mitochondria distribution, cells were transfected with the mitochondrial marker DsRed2-Mito with wt or mutant (P182L) HA-tagged HSPB1. All transfection and immunocytochemisty methods were as described earlier except that the primary antibody directed against the HA tag was detected using goat anti-rabbit Igs coupled to Alexa Fluor 488 (Molecular probes) with mitochondria being visualized using the Discosoma red fluorescent protein. Mitochondrial distribution was measured using a modified version of a previously described method (20Go). Briefly, images from transfected cells (not less than 50 cells) were captured using the same exposure time, a defined region of axon 100 µm in length beginning 50 µm from the cell body was then identified and the number of mitochondria present within this region counted. Statistical analyses of mitochondrial distribution were performed using Student's t-test.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Medical Research Council (K.T.), Jennifer Trust for SMA (S.A.) and Nuffield Medical Trust (P.J.). We would like to thank Vincent Timmerman, Chris Miller, Giampietro Schiavo and Kevin Vaughan for the kind gifts of plasmids.

Conflict of Interest statement. All authors wish to state that there are no conflicts of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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