Pharmacologic Activation of Aldehyde Metabolism to Protect Hematopoietic Stem Cells (HSC) in Murine Models of Fanconi Anemia (FA)

HSC loss in FA is due to failure to resolve DNA inter-strand crosslinks (ICL), which can be induced by reactive aldehydes, radiation, or other clastogenic agents. Aldehyde exposure may occur through environmental sources, e.g. ingestion, absorption, and inhalation, or endogenously as a byproduct of cellular metabolism. The ALDH2*2 genotype, a dominant-negative point mutation in the aldehyde dehydrogenase 2 (ALDH2) gene, causes the "Asian flushing syndrome" when ethanol (EtOH) is ingested, due to decreased metabolism of small aldehydes, particularly acetaldehyde. ALDH2*2 is a disease modifier in FA, causing more rapid bone marrow failure and earlier leukemia onset in doubly affected children. Similarly, mice experimentally doubly knocked out for FANCD2 and ALDH2 demonstrate increased HSC loss, which is accelerated by EtOH exposure. To reduce aldehyde exposure, we developed a small molecule ALDH activator, Alda-1, which increases the enzymatic activity of both wild type (WT) and mutant ALDH2. We hypothesized that DNA damage and HSC loss in FA would be prevented by reduction of the aldehyde load. To test the effects of Alda-1 mediated ALDH2 activation, we generated a novel murine FA model with FANCD2 KO and knock-in of the ALDH2*2 mutation into the murine locus. The FANCD2-/- ALDH2*1/*2 genetic model and parental controls were then tested after exogenous aldehydic challenge and/or therapeutic intervention with Alda-1. Increased aldehydic load was experimentally induced by EtOH administration 10 mg/kg/day IP, while Alda-1 10 ug/kg/day was continuously administered via osmotic pump. For each of these conditions, marrow was analyzed for HSC and progenitor cell (HSPC) number, HSC gene expression, and function. The importance of the altered aldehyde metabolism due to ALDH2*2 genotype was demonstrated by progressive loss of HSPC in ALDH2*2/*2 and FANCD2-/- ALDH2*1/*2 mice, e.g., 5-fold and 2-fold decline in long-term HSC (LT-HSC), respectively, by 36 weeks. Experimental EtOH challenge to increase the aldehyde load precipitously decreased HSC numbers of all genotypes. After 5 weeks of EtOH challenge, LT-HSC decreased 35-fold in FANCD2-/- ALDH2*1/*2, 12.5-fold in FANCD2-/-ALDH2*1/*1, and 10.5-fold in WT mice. Long-term Alda-1 treatment to decrease aldehydic load rescued FA mice from HSC loss. After 7 months of Alda-1 treatment, LT-HSC numbers in FANCD2-/-ALDH2*1/*2 and FANCD2-/-ALDH2*1/*1 were approximately 10-fold higher than untreated controls. There were no clinically observed adverse effects. Aldehyde exposure and Alda-1 treatment altered gene expression of HSC. Principal component analysis and clustering of HSC gene expression showed that the first principal component representing 40% of the variation in gene expression could be attributed to increased aldehydic load, either genetically (ALDH2*2 genotype) or environmentally (EtOH administration) induced, while Alda-1 treatment obviated these effects. HSC from Alda-1 treated mice clustered with those from control WT mice. To test whether Alda-1 improved HSC function as well as phenotypic number, engraftment potential was assessed with competitive repopulation assays of sorted HSC from congenic untreated donors vs short-term (3 weeks) Alda-1 treated donors. HSC from Alda-1 treated mice had 2-4 fold greater granulocyte repopulating capacity than those from the untreated donors. Our results demonstrate that Alda-1 treatment rescues HSC from aldehyde induced loss, whether from genetic variation (FANCD2- and/or ALDH2*2) or experimental challenge (EtOH administration). Furthermore, Alda-1 treatment prevents the abnormal HSC gene expression induced by increased aldehydic load. HSC function is improved by Alda-1 with greater repopulating capacity observed even after short-term treatment. These preclinical experiments provide compelling proof-of-concept that sustained activation of ALDH2 can prevent HSC loss in FA. Potential applications include long-term administration to prevent the development of marrow failure or leukemia, and HSC expansion to increase the number of cells available for gene therapy with autologous HSC. Our results suggest that a clinical trial of ALDH2 activation in FA patients to prevent marrow failure is warranted. Disclosures Van Wassenhove: U.S. Patent Office: Patents &amp; Royalties: patent pending - submitted for ALDH2 activators to expand hematopoietic stem cells. Chen:Foresee Pharmaceuticals: Patents &amp; Royalties: patents licensed to Foresee related to compounds that activate aldehyde dehydrogenase 2 (ALDH2) and correct the dysfunction in ALDH2*2; U.S. Patent Office: Patents &amp; Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Mochly-Rosen:Foresee Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties: patents licensed to Foresee related to compounds that activate aldehyde dehydrogenase 2 (ALDH2) and correct the dysfunction in ALDH2*2; U.S. Patent Office: Patents &amp; Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. Weinberg:U.S. Patent Office: Patents &amp; Royalties: patent pending - submitted for aldehyde dehydrogenase 2 (ALDH2) activators to expand hematopoietic stem cells. </jats:sec

Aldehyde dehydrogenase 2 activity and aldehydic load contribute to neuroinflammation and Alzheimer’s disease related pathology

AbstractAldehyde dehydrogenase 2 deficiency (ALDH2*2) causes facial flushing in response to alcohol consumption in approximately 560 million East Asians. Recent meta-analysis demonstrated the potential link between ALDH2*2 mutation and Alzheimer’s Disease (AD). Other studies have linked chronic alcohol consumption as a risk factor for AD. In the present study, we show that fibroblasts of an AD patient that also has an ALDH2*2 mutation or overexpression of ALDH2*2 in fibroblasts derived from AD patients harboring ApoE ε4 allele exhibited increased aldehydic load, oxidative stress, and increased mitochondrial dysfunction relative to healthy subjects and exposure to ethanol exacerbated these dysfunctions. In an in vivo model, daily exposure of WT mice to ethanol for 11 weeks resulted in mitochondrial dysfunction, oxidative stress and increased aldehyde levels in their brains and these pathologies were greater in ALDH2*2/*2 (homozygous) mice. Following chronic ethanol exposure, the levels of the AD-associated protein, amyloid-β, and neuroinflammation were higher in the brains of the ALDH2*2/*2 mice relative to WT. Cultured primary cortical neurons of ALDH2*2/*2 mice showed increased sensitivity to ethanol and there was a greater activation of their primary astrocytes relative to the responses of neurons or astrocytes from the WT mice. Importantly, an activator of ALDH2 and ALDH2*2, Alda-1, blunted the ethanol-induced increases in Aβ, and the neuroinflammation in vitro and in vivo. These data indicate that impairment in the metabolism of aldehydes, and specifically ethanol-derived acetaldehyde, is a contributor to AD associated pathology and highlights the likely risk of alcohol consumption in the general population and especially in East Asians that carry ALDH2*2 mutation.</jats:p

Development of an owner-assessed scale to measure the severity of pruritus in dogs

Assessment of pruritus is critical in dermatological history taking, both for initial diagnosis and for monitoring treatment. Various methods of assessing pruritus in the dog have been described but none have been validated. In this study, four different pruritus scales were initially assessed: a 0–5 numerical scale; a scale containing six categories of pruritic behaviours; a scale with six basic descriptors of severity; and a visual analogue scale with severity descriptions at either end. One hundred and sixteen owners were asked to grade their dog's pruritus with each of the four scales given to them in a randomized order, and asked which they thought was easiest to use, which was most accurate in assessing their dog's pruritus level, and which two scales they would combine to make a superior scale. Pruritus scores from different scales were highly correlated with each other, but scores were significantly higher with the severity and numerical scales compared to the behaviour scale. Based on the clients’ responses, a new scale was developed that combined features of the behaviour and severity-based scales with the visual analogue scale. When tested on 166 different owners, 98% thought it was easy to use and accurate in assessing their dog's pruritus level. In 44 dogs, independent scores obtained by two owners were highly correlated (R = 0.8; P < 0.0001), confirming the scale's repeatability. The new scale proved to be an easy and repeatable method for owners to determine the severity of pruritus in their dog.P. B. Hill, P. Lau and J. Rybnice