Knockdown of Heat Shock Proteins HSPA6 (Hsp70B') and HSPA1A (Hsp70-1) Sensitizes Differentiated Human Neuronal Cells to Cellular Stress.

Heat shock proteins are involved in cellular repair and protective mechanisms that counter characteristic features of neurodegenerative diseases such as protein misfolding and aggregation. The HSPA (Hsp70) multigene family includes the widely studied HSPA1A (Hsp70-1) and the little studied HSPA6 (Hsp70B') which is present in the human genome and not in mouse and rat. The effect of knockdown of HSPA6 and HSPA1A expression was examined in relation to the ability of differentiated human SH-SY5Y neuronal cells to tolerate thermal stress. Low dose co-application of celastrol and arimoclomol, which induces Hsps, enhanced the ability of differentiated neurons to survive heat shock. Small interfering RNA (siRNA) knockdown of HSPA6 and HSPA1A resulted in loss of the protective effect of co-application of celastrol/arimoclomol. More pronounced effects on neuronal viability were apparent at 44 °C heat shock compared to 43 °C. siRNA knockdown suggests that HSPA6 and HSPA1A contribute to protection of differentiated human neuronal cells from cellular stress.

Targeting of Heat Shock Protein HSPA6 (HSP70B') to the Periphery of Nuclear Speckles is Disrupted by a Transcription Inhibitor Following Thermal Stress in Human Neuronal Cells.

Heat shock proteins (Hsps) are a set of highly conserved proteins involved in cellular repair and protective mechanisms. The intracellular localization of inducible members of the HSPA (HSP70) family can be used as an index to identify stress-sensitive sites in differentiated human neuronal cells. Following thermal stress, the little studied HSPA6 (HSP70B') was targeted to the periphery of nuclear speckles (perispeckles) that are sites of transcription factories. Triptolide, a fast-acting transcription inhibitor, knocked down levels of the large subunit of RNA polymerase II, RPB1, during the time-frame when HSPA6 associated with perispeckles. Administration of triptolide to heat shocked human neuronal SH-SY5Y cells, disrupted HSPA6 localization to perispeckles, suggesting the involvement of HSPA6 in transcriptional recovery after stress. The HSPA6 gene is present in the human genome but is not found in the genomes of the mouse and rat. Hence current animal models of neurodegenerative diseases lack a member of the HSPA family that exhibits the feature of stress-induced targeting to perispeckles.

Intracellular Targeting of Heat Shock Proteins in Differentiated Human Neuronal Cells Following Proteotoxic Stress.

HSPA6 (Hsp70B') is an inducible member of the Hsp70 (HSPA) family of heat shock proteins that is present in the human genome and not found in mouse and rat. Hence it is lacking in current animal models of neurodegenerative diseases. To advance knowledge of the little studied HSPA6, differentiated human neuronal SH-SY5Y cells were treated with the proteotoxic stress-inducing agent MG132. A robust induction of HSPA6 was apparent which localized to the periphery of MG132-induced protein aggregates in the neuronal cytoplasm. Components of the protein disaggregation/refolding machine that co-operate with Hsp70 also targeted the periphery of cytoplasmic protein aggregates, including DNAJB1 (Hsp40-1), HSPH1 (Hsp105α), and HSPB1 (Hsp27). These data suggest that HSPA6 is involved in the response of human neuronal cells to proteotoxic stress that is a feature of neurodegenerative diseases which have been characterized as protein misfolding disorders. Constitutively expressed HSPA8 (Hsc70) also localized tothe periphery of cytoplasmic protein aggregates following the treatment of differentiated human neuronal cells with MG132. HSPA8 could provide a rapid response to proteotoxic stress in neuronal cells, circumventing the time required to upregulate inducible Hsps.

Differential Targeting of Hsp70 Heat Shock Proteins HSPA6 and HSPA1A with Components of a Protein Disaggregation/Refolding Machine in Differentiated Human Neuronal Cells following Thermal Stress.

Heat shock proteins (Hsps) co-operate in multi-protein machines that counter protein misfolding and aggregation and involve DNAJ (Hsp40), HSPA (Hsp70), and HSPH (Hsp105α). The HSPA family is a multigene family composed of inducible and constitutively expressed members. Inducible HSPA6 (Hsp70B') is found in the human genome but not in the genomes of mouse and rat. To advance knowledge of this little studied HSPA member, the targeting of HSPA6 to stress-sensitive neuronal sites with components of a disaggregation/refolding machine was investigated following thermal stress. HSPA6 targeted the periphery of nuclear speckles (perispeckles) that have been characterized as sites of transcription. However, HSPA6 did not co-localize at perispeckles with DNAJB1 (Hsp40-1) or HSPH1 (Hsp105α). At 3 h after heat shock, HSPA6 co-localized with these members of the disaggregation/refolding machine at the granular component (GC) of the nucleolus. Inducible HSPA1A (Hsp70-1) and constitutively expressed HSPA8 (Hsc70) co-localized at nuclear speckles with components of the machine immediately after heat shock, and at the GC layer of the nucleolus at 1 h with DNAJA1 and BAG-1. These results suggest that HSPA6 exhibits targeting features that are not apparent for HSPA1A and HSPA8.

Components of a mammalian protein disaggregation/refolding machine are targeted to nuclear speckles following thermal stress in differentiated human neuronal cells.

Heat shock proteins (Hsps) are a set of highly conserved proteins involved in cellular repair and protective mechanisms. They counter protein misfolding and aggregation that are characteristic features of neurodegenerative diseases. Hsps act co-operatively in disaggregation/refolding machines that assemble at sites of protein misfolding and aggregation. Members of the DNAJ (Hsp40) family act as "holdases" that detect and bind misfolded proteins, while members of the HSPA (Hsp70) family act as "foldases" that refold proteins to biologically active states. HSPH1 (Hsp105α) is an important additional member of the mammalian disaggregation/refolding machine that acts as a disaggregase to promote the dissociation of aggregated proteins. Components of a disaggregation/refolding machine were targeted to nuclear speckles after thermal stress in differentiated human neuronal SH-SY5Y cells, namely: HSPA1A (Hsp70-1), DNAJB1 (Hsp40-1), DNAJA1 (Hsp40-4), and HSPH1 (Hsp105α). Nuclear speckles are rich in RNA splicing factors, and heat shock disrupts RNA splicing which recovers after stressful stimuli. Interestingly, constitutively expressed HSPA8 (Hsc70) was also targeted to nuclear speckles after heat shock with elements of a disaggregation/refolding machine. Hence, neurons have the potential to rapidly assemble a disaggregation/refolding machine after cellular stress using constitutively expressed Hsc70 without the time lag needed for synthesis of stress-inducible Hsp70. Constitutive Hsc70 is abundant in neurons in the mammalian brain and has been proposed to play a role in pre-protecting neurons from cellular stress.