Roles Of Mitochondria Affected By The K141N Mutant Of HSP22in The Pathogenesis Of CMT2L

Backgroud:Charcot-Marie-Tooth disease (CMT) was first reported by Charcot, Marie, Tooth in1886, which is the most common inherited peripheral neuropathy, the incidence rate is about1/2500. According to the pathological and electrophysiological characteristics CMT can be divided into three types:demyelinating form (CMT1type), axonal form (CMT2type) and intermediate form (ICMT type). So far, more than40different CMT loci have been identified, approximately30genes have been cloned. Point mutations (423Gâ†'T) in small heat shock protein22(HSP22) gene was shown to cause CMT2L. Several CMT2pathogenic genes can change the mitochondrial dynamics, mitochondrial energy metabolism or mitochondrial axoplasmic transport, such as heat shock protein (HSP27), neurofilament light chain (NEFL), ganglioside-induced differentiation-related protein1(GDAP1), mitochondrial fusion protein2(MFN2), kinesin family member1B (KIF1B) and dehydrogenase El and transketolase domain-containing1(DHTKD1). Also, Irobi studied the effect of mutant HSP22in primary fibroblast cultrues derived from dHMNII patient’s skin biopsy, found a reduction in mitochondrial membrane potential. We hypothesized that the mutant HSP22might affect mitochondria in CMT2LObjective:To study weather the mutant HSP22can affect mitochondria, we detected mitochondrial distribution by immunofluorescence techniques, and mitochondrial membrane potential was observed by flow cytometry.Methods:(1)Observe mitochondrial distribution affected by the mutant HSP22using immunofluorescence techniques.(2) Detect mitochondrial membrane potential by flow cytometry.(3) Study the effect of the mutant HSP22to mitochondria in spinal motor neurons infected with lentivirus by immunofluorescence techniques.Results:(1) We transfected pLVX-Puro-EGFP-HSP22WT, pLVX-Puro-EGFP-HSP22MT plasmid into SHSY5Y cells and observed mitochondria using immunofluorescence technique. No abnormality was found in the wild type group, mitochondria in the mutant type group showed abnormal accumulation and colocalization with mutant HSP22.(2) We transfected pLVX-Puro-EGFP-HSP22WT, pLVX-Puro-EGFP-HSP22MT plasmid into HEK293cells and observed mitochondria using immunofluorescence technique. No abnormality was found in the wild type group, but the K141N mutant HSP22protein formed aggregates in the cell. No co-localization was shown between HSP22and mitochondria.(3) We transfected pLVX-Puro-EGFP-HSP22WT, pLVX-Puro-EGFP-HSP22MT plasmid into SHSY5Y cells and detected mitochondrial membrane potential by flow cytometry. No significant difference of mitochondrial membrane potential was detected between control group and the mutant type group. To further investigate the influence of mutant HSP22to mitochondrial membrane potential in stress, we treated the cells with CCCP for6hours. Both the wild type group and the mutant type group presented a reduction of mitochondrial membrane potential. The mitochondrial membrane potential of mutant type group was lower than that of the positive control group.(4) We detected mitochondrial distribution in spinal motor neurons infected with lentivirus. The wild type group showed an evenly distribution of mitochondria throughout the neuronal cell bodies and axons. There was no abnormal accumulation of the HSP22protein; the mutant type group showed mitochondria aggregation in axon and co-localization with mutant HSP22accumulation.Conclusions:(1) Mitochondria in mutant type cells originated from nervous system showed abnormal accumulation and colocalization with mutant HSP22.(2) The K141N mutant of HSP22presented no toxicity effect on mitochondrial membrane potential, but showed a loss of protective function during stress.(3)The mutant group showed mitochondrial aggregation in axon and co-localization with mutant HSP22accumulation, suggesting abnormal axoplasmic transport of mitochondria.