<i>Fkbp5</i> DNA methylation decreases with age in mice.

<p>Isolated DNA samples from wild-type mice aged 1 (n = 3), 3.5 (n = 3), 4 (n = 6), 5 (n = 7), 6 (n = 6), and 12 (n = 3) months were subjected to bisulfite pyrosequencing. Multiple CpG sites in intron 5 were analyzed for <i>Fkbp5</i> methylation. Significant demethylation was found at CPG_3 (r = −0.3890, p<0.05), CPG_4 (r = −0.4004, p<0.05) and CpG_5 (r = −0.5044, p<0.01) as measured by linear regression analyses.</p

<i>FKBP5^−/−</i> mice display enhanced cognitive flexibility in the radial arm water maze.

<p>(A) No differences were found in acquisition learning between genotypes (p>0.05). (B) There was a main effect of genotype (p<0.05) during reversal training, indicating <i>FKBP5^−/−</i> mice made fewer errors across sessions. Data points represent a session of three trials. *p<0.05. wild-type (wt), n = 9; <i>FKBP5^−/−</i>, n = 10.</p

FKBP51 does not affect longevity.

<p>No significant differences were found in the percent survival of wild-type (wt) and <i>FKBP5^−/−</i> mice, p>0.05. wt, n = 34 (18 male and 16 female); <i>FKBP5^−/−</i>, n = 32 (18 male and 14 female).</p

Deletion of <i>FKBP5</i> does not alter glucose metabolism.

<p><i>FKBP5^−/−</i> mice displayed normal glucose tolerance up to 120 minutes following glucose injection compared to wild-type (wt) mice, p>0.05. wt, n = 7; <i>FKBP5^−/−</i>, n = 10.</p

Ablation of <i>FKBP5</i> does not alter cytokine levels over time.

<p>Serum levels of interleukin-1β (A) and interleukin-5 (D) were decreased at 6 months (p<0.05 via t-test) but not across time (p>0.05 by two-way ANOVA). Levels of interleukin-2 (B), interleukin-4 (C), interleukin-10 (E), granulocyte-macrophage colony-stimulating factor (F), interferon gamma (G), or tumor necrosis factor alpha (H) did not differ between genotypes across lifespan, p>0.05. *p<0.05. wild-type (wt), n = 6 for each age; <i>FKBP5^−/−</i>, n = 7 at 7 and 10 months, n = 6 at 21 months.</p

Blood composition and animal mass of wild-type versus FKBP5^−/− mice do not differ.

<p>WBC: white blood cells, RBC: red blood cells, Lym: lymphocytes, Mono: monocytes, Gran: granulocytes, HGB: hemoglobin, PLT: platelets. Wild-type, n = 11; FKBP5^−/−, n = 12. Values are listed as the mean ± the standard error of the mean.</p><p>Blood composition and animal mass of wild-type versus FKBP5^−/− mice do not differ.</p

Inhibition of Both Hsp70 Activity and Tau Aggregation <i>in Vitro</i> Best Predicts Tau Lowering Activity of Small Molecules

Three scaffolds with inhibitory activity against the heat shock protein 70 (Hsp70) family of chaperones have been found to enhance the degradation of the microtubule associated protein tau in cells, neurons, and brain tissue. This is important because tau accumulation is linked to neurodegenerative diseases including Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE). Here, we expanded upon this study to investigate the anti-tau efficacy of additional scaffolds with Hsp70 inhibitory activity. Five of the nine scaffolds tested lowered tau levels, with the rhodacyanine and phenothiazine scaffolds exhibiting the highest potency as previously described. Because phenothiazines also inhibit tau aggregation <i>in vitro</i>, we suspected that this activity might be a more accurate predictor of tau lowering. Interestingly, the rhodacyanines did inhibit <i>in vitro</i> tau aggregation to a similar degree as phenothiazines, correlating well with tau-lowering efficacy in cells and <i>ex vivo</i> slices. Moreover, other Hsp70 inhibitor scaffolds with weaker tau-lowering activity in cells inhibited tau aggregation <i>in vitro</i>, albeit at lower potencies. When we tested six well-characterized tau aggregation inhibitors, we determined that this mechanism of action was not a better predictor of tau-lowering than Hsp70 inhibition. Instead, we found that compounds possessing both activities were the most effective at promoting tau clearance. Moreover, cytotoxicity and PAINS activity are critical factors that can lead to false-positive lead identification. Strategies designed around these principles will likely yield more efficacious tau-lowering compounds

Human cyclophilin 40 unravels neurotoxic amyloids

<div><p>The accumulation of amyloidogenic proteins is a pathological hallmark of neurodegenerative disorders. The aberrant accumulation of the microtubule associating protein tau (MAPT, tau) into toxic oligomers and amyloid deposits is a primary pathology in tauopathies, the most common of which is Alzheimer’s disease (AD). Intrinsically disordered proteins, like tau, are enriched with proline residues that regulate both secondary structure and aggregation propensity. The orientation of proline residues is regulated by cis/trans peptidyl-prolyl isomerases (PPIases). Here we show that cyclophilin 40 (CyP40), a PPIase, dissolves tau amyloids in vitro. Additionally, CyP40 ameliorated silver-positive and oligomeric tau species in a mouse model of tau accumulation, preserving neuronal health and cognition. Nuclear magnetic resonance (NMR) revealed that CyP40 interacts with tau at sites rich in proline residues. CyP40 was also able to interact with and disaggregate other aggregating proteins that contain prolines. Moreover, CyP40 lacking PPIase activity prevented its capacity for disaggregation in vitro. Finally, we describe a unique structural property of CyP40 that may permit disaggregation to occur in an energy-independent manner. This study identifies a novel human protein disaggregase and, for the first time, demonstrates its capacity to dissolve intracellular amyloids.</p></div

Cyclophilin 40 (CyP40) overexpression decreased sarkosyl-insoluble tau.

<p>(A) Schematic depicting the timeline of rTg4510 tau pathology and experimental design. (B) Western blot comparing sarkosyl-insoluble and sarkosyl-soluble fractions from the hippocampi of adeno-associated virus serotype 9–green fluorescent protein (AAV9-GFP) (<i>n</i> = 8) and AAV9-CyP40 (<i>n</i> = 8) injected rTg4510 mice. Each lane indicates an individual transgenic mouse. (C) Quantification of the relative insoluble and soluble tau for AAV9-GFP and AAV9-CyP40 injected mice (unpaired <i>t</i> test, <i>n</i> = 8 for each group). The numerical data used in panel 2C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001336#pbio.2001336.s012" target="_blank">S1 Data</a>. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, standard error of the mean.</p

Cyclophilin 40 (CyP40) binds proline-containing regions of tau and α-synuclein (α-syn).

<p>(A) A selected region of the 2D [¹H-¹⁵N]–heteronuclear single quantum coherence (HSQC) experiment of ¹⁵N-labeled α-syn before (blue) and after (red) addition of CyP40 (molar ratio 1:15). Y136 is marked. (B) Normalized residue-specific nuclear magnetic resonance (NMR) intensities in the presence of a 5-fold (red) and 15-fold (blue) excess of Cyp40; the location of proline residues is indicated. (C) Combined [¹H-¹⁵N] chemical shift perturbation analysis upon addition of CyP40 to α-syn. (D) A selected region of 2D [¹H-¹⁵N]-HSQC experiment of tau before (blue) and after (red) addition of CyP40 (molar ratio 1:10) is shown. Residues displaying significant intensity loss are labeled. (E) Normalized residue-specific NMR intensities of tau in the presence of a 5-fold (red) and 10-fold (blue) excess of CyP40; tau’s domain organization is shown, and proline residues are marked with red. (F) Amino acid sequence of the proline-rich regions P1 (top) and P2 (bottom); residues significantly broadened upon addition of CyP40 are colored red. (G) A selected region of the 2D [¹H-¹⁵N]-HSQC experiment of ¹⁵N-labeled α-syn before (blue) and after (red) addition of FK-506 binding protein (FKBP) 51 (molar ratio 1:10) is displayed. (H) Normalized residue-specific NMR intensities in the presence of a 5-fold (red) and 10-fold (blue) excess of FKBP51. (I) Combined [¹H-¹⁵N] chemical shift perturbation analysis upon addition of FKBP51 to α-syn. (J) Selected region of a 2D [¹H-¹⁵N]-HSQC experiment of tau before (blue) and after (red) addition of FKBP51 (molar ratio 1:10). Final concentrations of CyP40 and FKBP51 for NMR experiments are 200 μM. (K) Tau residue-specific NMR intensities in presence of a 10-fold excess of FKBP51. (L) Thioflavin T fluorescence of K18 tau aggregates in the presence or absence of CyP40 (unpaired <i>t</i> test, <i>p</i> > 0.05, <i>n</i> = 3). The numerical data used in panel 6L can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001336#pbio.2001336.s012" target="_blank">S1 Data</a>.</p