Low-dose Carfilzomib in <i>Tg-S466L</i> mice.

Mice were treated at a dose of 2.1 mg/Kg/day for one week using an osmotic pump. A. Comparison of tHcy in untreated (UT) and treated (LD Carf) mice. B. Liver CBS activity. C. Western blot analysis.</p

Long-term, low-dose bortezomib in <i>Tg-I278T</i> mice.

A. Diagram showing injection and blood collection times. Injections amounts and times are shown above the line, blood and tissue collection is shown below. B. tHcy at indicated time. Significance was determined by as different from d0 time point. C. Weight of animals during experiment. D. CBS activity. Red circle shows points for animal that appeared moribund at time of sacrifice. E. Blood counts at end of experiment. WBC = white blood cells; LYM = lymphocytes; Mon = monocytes; Neu = neutrophils; RBC = red blood cells.</p

Notebook data for Western blots.

File shows complete image data for all Western blots, as well as molecular weight migration markers. (PDF)</p

Western blot analysis of humanized CBS mice treated with either nothing, carfilzomib or bortezomib.

Carfilzomib was administered as a single dose by R.O. injection at 10 mg/kg. Bortezomib was administered via osmotic pump delivering 0.49 mg/kg/day. Carfilzomib mice were euthanized 24 hours after injection, while bortezomib mice were euthanized 36–48 hours after implantation. A. Blot showing liver extracts from Tg-R266K mice. B. Liver extracts from Tg-R336C mice. C. Liver extracts from Tg-I278T mice. Red asterisk denotes highly responsive sample. D. Liver extracts from Tg-T191M mice. Lanes labeled hCBS contain extracts from Tg-hCBS mice [18].</p

Liver CBS activity and serum tHcy, in treated humanized CBS mice.

A. Liver CBS activity in indicated strains with indicated treatments. Red circle denotes highly responsive sample noted in Fig 2C. B. Serum tHcy levels in indicated strains with indicated treatments. Error bars show SEM. Asterisks indicate significant differences from untreated samples: *p<0.05, **p<0.01, ***p<0.001, and ****P<0.0001.</p

Underlying data for all graphs.

Classic homocystinuria is an inborn error of metabolism caused mainly by missense mutations leading to misfolded and/or unstable human cystathionine β-synthase (CBS) protein, causing the accumulation of excess total homocysteine (tHcy) in tissues. Previously, it has been shown that certain missense containing human CBS proteins can be functionally rescued in mouse models of CBS deficiency by treatment with proteasome inhibitors. The rescue by proteasome inhibitors is thought to work both by inhibiting the degradation of misfolded CBS protein and by inducing the levels of heat-shock chaperone proteins in the liver. Here we examine the effectiveness of two FDA approved protease inhibitors, carfilzomib and bortezomib, on various transgenic mouse models of human CBS deficiency. Our results show that although both drugs are effective in inducing the liver chaperone proteins Hsp70 and Hsp27, and are effective in inhibiting proteasome function, bortezomib was somewhat more robust in restoring the mutant CBS function. Moreover, there was no significant correlation between proteasome inhibition and CBS activity, suggesting that some of bortezomib’s effects are via other mechanisms. We also test the use of low-doses of bortezomib and carfilzomib on various mouse models for extended periods of time and find that while low-doses are less toxic, they are also less effective at restoring CBS function. Overall, these results show that while restoration of mutant CBS function is possible with proteasome inhibitors, the exact mechanism is complicated and it will likely be too toxic for long-term patient treatment.</div

Low-dose bortezomib combined with bimoclomol in <i>Tg-I278T</i> mice.

A. Treatment scheme. Injection days and amounts shown on top. B. tHcy pre and post treatment. C. Liver CBS enzyme activity at end of treatment. D. Western blot of hCBS and indicated Hsps. Note that two bimoclomol only treated animals (Bimo) are included as controls.</p

Proteasome activity of bortezomib- and carfilzomib-treated mice.

A. left side shows liver proteasome activity of Tg-R266K liver extracts expressed as percent of untreated control Tg-hCBS extract. Assay was performed using a fluorescent substrate. On right side % proteasome activity is plotted on X-axis vs. liver CBS activity on Y-axis. Color coding is: Black = untreated, red = carfilzomib-treated, blue = bortezomib-treated. B. Same as above for Tg-R336C. C. Same as above for Tg-I278T. D. Same as above for Tg-T191M.</p

Effect of carfilzomib on <i>Tg-S466L Cbs</i>^<i>-/-</i> mice.

A. Serum tHcy in treated and untreated mice. Ut = untreated, S.C. = subcutaneous delivery using an osmotic pump (15 mg/kg), R.O. = retro-orbital injection (10 mg/kg). For retro-orbital (R.O.) injected animals two time points were examined. Number of animals and standard error of mean (SEM) is shown. Asterisks indicate significant differences from untreated samples: *p<0.05, **p<0.01. B. Liver CBS activity of treated and untreated mice. C. Western blot analysis of R.O. and S.C. treated animals. Saline-treated control is shown on right. At top of panel, tHcy and liver CBS activity for each mouse is also shown.</p

The TSC1/2 Complex Controls <em>Drosophila</em> Pigmentation through TORC1-Dependent Regulation of Catecholamine Biosynthesis

<div><p>In <em>Drosophila</em>, the pattern of adult pigmentation is initiated during late pupal stages by the production of catecholamines DOPA and dopamine, which are converted to melanin. The pattern and degree of melanin deposition is controlled by the expression of genes such as <em>ebony</em> and <em>yellow</em> as well as by the enzymes involved in catecholamine biosynthesis. In this study, we show that the conserved TSC/TORC1 cell growth pathway controls catecholamine biosynthesis in <em>Drosophila</em> during pigmentation. We find that high levels of Rheb, an activator of the TORC1 complex, promote premature pigmentation in the mechanosensory bristles during pupal stages, and alter pigmentation in the cuticle of the adult fly. Disrupting either melanin synthesis by RNAi knockdown of melanogenic enzymes such as <em>tyrosine hydroxylase</em> (TH), or downregulating TORC1 activity by Raptor knockdown, suppresses the Rheb-dependent pigmentation phenotype in vivo. Increased Rheb activity drives pigmentation by increasing levels of TH in epidermal cells. Our findings indicate that control of pigmentation is linked to the cellular nutrient-sensing pathway by regulating levels of a critical enzyme in melanogenesis, providing further evidence that inappropriate activation of TORC1, a hallmark of the human tuberous sclerosis complex tumor syndrome disorder, can alter metabolic and differentiation pathways in unexpected ways.</p> </div