Hsp72 (HSPA1A) Prevents Human Islet Amyloid Polypeptide Aggregation and Toxicity: A New Approach for Type 2 Diabetes Treatment

Type 2 diabetes is a growing public health concern and accounts for approximately 90% of all the cases of diabetes. Besides insulin resistance, type 2 diabetes is characterized by a deficit in β-cell mass as a result of misfolded human islet amyloid polypeptide (h-IAPP) which forms toxic aggregates that destroy pancreatic β-cells. Heat shock proteins (HSP) play an important role in combating the unwanted self-association of unfolded proteins. We hypothesized that Hsp72 (HSPA1A) prevents h-IAPP aggregation and toxicity. In this study, we demonstrated that thermal stress significantly up-regulates the intracellular expression of Hsp72, and prevents h-IAPP toxicity against pancreatic β-cells. Moreover, Hsp72 (HSPA1A) overexpression in pancreatic β-cells ameliorates h-IAPP toxicity. To test the hypothesis that Hsp72 (HSPA1A) prevents aggregation and fibril formation, we established a novel C. elegans model that expresses the highly amyloidogenic human pro-IAPP (h-proIAPP) that is implicated in amyloid formation and β-cell toxicity. We demonstrated that h-proIAPP expression in body-wall muscles, pharynx and neurons adversely affects C. elegans development. In addition, we demonstrated that h-proIAPP forms insoluble aggregates and that the co-expression of h-Hsp72 in our h-proIAPP C. elegans model, increases h-proIAPP solubility. Furthermore, treatment of transgenic h-proIAPP C. elegans with ADAPT-232, known to induce the expression and release of Hsp72 (HSPA1A), significantly improved the growth retardation phenotype of transgenic worms. Taken together, this study identifies Hsp72 (HSPA1A) as a potential treatment to prevent β-cell mass decline in type 2 diabetic patients and establishes for the first time a novel in vivo model that can be used to select compounds that attenuate h-proIAPP aggregation and toxicity

Expression of h-proIAPP in <i>C</i>. <i>elegans</i> results in protein insolubility and aggregation.

<p>Transgenic h-proIAPP <i>C</i>. <i>elegans</i> model was generated by gonad microinjection of 20 ng/μl of plasmids pPR3, pPR4 and pPR5. Transgenic m-proIAPP <i>C</i>. <i>elegans</i> model was generated by gonad microinjection of 20 ng/μl of plasmids pPR8, pPR9 and pPR10. <b>A.</b> h-proIAPP and m-proIAPP tagged with YFP were expressed in body wall muscles, vulva muscles and anal depressor muscles under <i>lev-11</i> promoter; in pharynx under <i>tnt-4</i> promoter, and in neurons under <i>aex-3</i> promoter. Neuronal expression of proIAPP secreted into the body cavity was observed in the coelomocytes of the h-proIAPP model and distributed in the intestinal region in both h-proIAPP and m-proIAPP <i>C</i>. <i>elegans</i> models. Images were created using maximum intensity projections on all the planes of the acquired stacks except coelomocytes where images were taken in a single plane for better visualization. Arrows indicate areas of fluorescence. DIC images were taken to visualize structures. Results are a representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. <b>B.</b> Data represent the mean fluorescence intensity (MFI) ± SD measured using a sum intensity projection on all the planes of the acquired stacks of m-proIAPP (open bars) and h-proIAPP (filled bars) tagged with YFP expressed in body wall muscles, vulva muscles, anal depressor muscles and pharynx. *p<0.05; **p<0.01 versus m-proIAPP. <b>C.</b> Transgenic h-proIAPP <i>C</i>. <i>elegans</i> tagged with YFP were subjected to FRAP analysis (square). Data are from vulva muscles (row 1), anal depressor (row 2), pharynx (row 3) and coelomocytes (row 4) before FRAP (left panels), 10 seconds after FRAP (middle panels) 250 seconds after FRAP (right panels). Images were obtained using an inverted confocal microscope. Results are representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm.</p

Expression of h-proIAPP in <i>C</i>. <i>elegans</i> correlates with growth retardation phenotype.

<p>Developmental phenotype was studied in transgenic h-proIAPP <i>C</i>. <i>elegans</i> model, transgenic m-proIAPP <i>C</i>. <i>elegans</i> model and pBX1 control animals by quantifying the number of animals in the larvae 1 (L1) to larvae 3 (L3) stages (open bars) versus larvae 4 (L4) and adult stages (filled bars) seventy-two hours after ten to fifteen gravid hermaphrodites were treated with bleach to release eggs. Data represent the percentage of animals found at larvae 1 to larvae 3 stages and the percentage of animals found at larvae 4 and adult stages ± S.D. p<0.05 versus respective control, n = 3.</p

Hsp72 expression improves the solubility of h-proIAPP aggregates.

<p><b>A.</b> Transgenic YFP + Hsp72 <i>C</i>. <i>elegans</i> model was generated by co-injection of 20 ng/μl of plasmid pPR18 (that expresses YFP in muscles) together with 30 ng/μl of Hsp72 expressed in the same tissue (plasmid pPR21) to serve as a soluble control (top panels). <i>C</i>. <i>elegans</i> injected with 20 ng/μl of h-proIAPP tagged with YFP expressed in muscles (plasmid pPR3) was used as a control for protein aggregation (middle panels). Transgenic h-proIAPP::YFP + Hsp72 <i>C</i>. <i>elegans</i> animals were generated by gonad co-injection of 20 ng/μl of plasmid pPR3 and 30 ng/μl of plasmid pPR21 (bottom panels). Transgenic animals were subjected to FRAP analysis (square). Data was collected before photobleaching (left panels), 10 seconds after photobleaching (middle panels) and 250 seconds after photobleaching (right panels). Images were obtained using an inverted confocal microscope. Results are representative of one experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. <b>B.</b> Data represents the quantification of relative fluorescence intensity, RFI ± SEM during recovery after photobleaching of transgenic h-proIAPP::YFP <i>C</i>. <i>elegans</i> model (filled circles), transgenic h-proIAPP::YFP + Hsp72 <i>C</i>. <i>elegans</i> animals (open circles), and transgenic YFP + Hsp72 <i>C</i>. <i>elegans</i> model (filled triangles). Data are the mean of at least three independently performed experiments. *p<0.05 versus h-proIAPP::YFP, Error Bar = SEM.</p

Construction and identification of human and mouse proIAPP <i>C</i>. <i>elegans</i> vectors.

<p><b>A.</b> Schematic representation of human-proIAPP and mouse-proIAPP tissue-specific vector constructs. cDNA of human and mouse proIAPP with human and mouse signal peptide, respectively, were cloned into the BamH1 restriction site of the pSX95.77YFP <i>C</i>.<i>elegans</i> vector to generate human and mouse pSX95.77YFP-proIAPP transgenes. Resulting constructs were digested with Sal1, blunt-ended and ligated into Gateway Cassette C.1 upstream of human and mouse coding sequences to create vectors pNG1 and pNG2. Destination vectors pNG1 and pNG2 were LR recombined with pLR22, pLR25 and pLR35 entry clones to generate human proIAPP plasmids: pPR3, pPR4 and pPR5 and mouse proIAPP plasmids: pPR8, pPR9 and pPR10. <b>B.</b> h-proIAPP sequence of construct pSX95.77YFP-prohIAPP with human signal peptide. Preproh-IAPP (1–89) peptide amino acid sequence is shown in black italics. Signal peptide sequence is shown in red. <b>C.</b> m-proIAPP sequence present in construct pSX95.77YFP-promIAPP with mouse signal peptide. Preprom-IAPP peptide amino acid sequence is shown in black italics. Signal peptide sequence is shown in red. Restriction sites used in the generation of these plasmids are underlined. No stop codon was included after each proIAPP sequence.</p

Hsp72 reduces endogenous h-IAPP toxicity in pancreatic β-cells.

<p><b>A.</b> Beta-TC-6 cells were transfected and cell viability was assessed by MTS assay. Data represent percentage of live cells ± SD (filled bars) when transfected with vector pCMV6-XL5 (control) or human Hsp72::EGFP cDNA clone or m-proIAPP cDNA clone or h-proIAPP cDNA clone or both h-proIAPP and human Hsp72::EGFP vectors. Data represents the sum of three independently performed experiments. **p<0.01 versus respective controls, n = 3. <b>B.</b> At the end of the transfection period, phase contrast and fluorescence images were obtained. Data is a representative experiment from at least three independently performed experiments with similar results. Scale bars represent 50 μm. <b>C.</b> Beta-TC-6 cells were transfected with empty vector pCMV6-XL5 (control), or m-proIAPP cDNA clone or h-proIAPP cDNA clone or with both h-proIAPP and human Hsp72::EGFP vectors. After transfection, samples were collected and examined for the expression of h-IAPP by Western blot analysis. Data is a representative experiment from at least three independently performed experiments with similar results. <b>D.</b> Beta-TC-6 cells were transfected with empty vector pCMV6-XL5 (control), or human Hsp72::EGFP cDNA clone, or with both h-proIAPP and human Hsp72::EGFP vectors. After transfection, samples were collected and examined for the expression of Hsp72 by Western blot analysis. Data is a representative experiment from at least three independently performed experiments with similar results.</p

HCN4 provides a ‘depolarization reserve' and is not required for heart rate acceleration in mice

Cardiac pacemaking involves a variety of ion channels, but their relative importance is controversial and remains to be determined. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which underlie the If current of sinoatrial cells, are thought to be key players in cardiac automaticity. In addition, the increase in heart rate following beta-adrenergic stimulation has been attributed to the cAMP-mediated enhancement of HCN channel activity. We have now studied mice in which the predominant sinoatrial HCN channel isoform HCN4 was deleted in a temporally controlled manner. Here, we show that deletion of HCN4 in adult mice eliminates most of sinoatrial If and results in a cardiac arrhythmia characterized by recurrent sinus pauses. However, the mutants show no impairment in heart rate acceleration during sympathetic stimulation. Our results reveal that unexpectedly the channel does not play a role for the increase of the heart rate; however, HCN4 is necessary for maintaining a stable cardiac rhythm, especially during the transition from stimulated to basal cardiac states