Triheptanoin protects motor neurons and delays the onset of motor symptoms in a mouse model of amyotrophic lateral sclerosis

There is increasing evidence that energy metabolism is disturbed in Amyotrophic Lateral Sclerosis (ALS) patients and animal models. Treatment with triheptanoin, the triglyceride of heptanoate, is a promising approach to provide alternative fuel to improve oxidative phosphorylation and aid ATP generation. Heptanoate can be metabolized to propionyl-CoA, which after carboxylation can produce succinyl-CoA and thereby re-fill the tricarboxylic acid (TCA) cycle (anaplerosis). Here we tested the hypothesis that treatment with triheptanoin prevents motor neuron loss and delays the onset of disease symptoms in female mice over-expressing the mutant human SOD1(G93A) (hSOD1(G93A)) gene. When oral triheptanoin (35% of caloric content) was initiated at P35, motor neuron loss at 70 days of age was attenuated by 33%. In untreated hSOD1(G93A) mice, the loss of hind limb grip strength began at 16.7 weeks. Triheptanoin maintained hind limb grip strength for 2.8 weeks longer (

Oxaliplatin-induced haematological toxicity and splenomegaly in mice.

PurposeHaematological toxicities occur in patients receiving oxaliplatin. Mild anaemia (grade 1-2) is a common side effect and approximately 90% of recipients develop measurable spleen enlargement. Although generally asymptomatic, oxaliplatin-induced splenomegaly is independently associated with complications following liver resection for colorectal liver metastasis and separately with poorer patient outcomes. Here, we investigated oxaliplatin-induced haematological toxicities and splenomegaly in mice treated with escalating dosages comparable to those prescribed to colorectal cancer patients.MethodsBlood was analysed, and smears assessed using Wright-Giemsa staining. Paw coloration was quantified as a marker of anaemia. Spleen weight and morphology were assessed for abnormalities relating to splenomegaly and a flow cytometry and multiplex cytokine array assessment was performed on splenocytes. The liver was assessed for sinusoidal obstructive syndrome.ResultsBlood analysis showed dose dependent decreases in white and red blood cell counts, and significant changes in haematological indices. Front and hind paws exhibited dose dependent and dramatic discoloration indicative of anaemia. Spleen weight was significantly increased indicating splenomegaly, and red pulp tissue exhibited substantial dysplasia. Cytokines and chemokines within the spleen were significantly affected with temporal upregulation of IL-6, IL-1α and G-CSF and downregulation of IL-1β, IL-12p40, MIP-1β, IL-2 and RANTES. Flow cytometric analysis demonstrated alterations in splenocyte populations, including a significant reduction in CD45+ cells. Histological staining of the liver showed no evidence of sinusoidal obstructive syndrome but there were signs suggestive of extramedullary haematopoiesis.ConclusionChronic oxaliplatin treatment dose dependently induced haematological toxicity and splenomegaly characterised by numerous physiological and morphological changes, which occurred independently of sinusoidal obstructive syndrome

Lower maximal activities of 2-oxoglutarate dehydrogenase (OGDH) in hSOD1^G93A gastrocnemius muscle.

<p>The maximal activities of OGDH in extracts from gastrocnemius muscle of male wild-type and hSOD1^G93A mice at different disease stages are compared. Stages are defined as presymptomatic (days 35–36), onset (days 63–75), mid-stage (days 110–130) and end-stage (days 150–175). The inset above the graph shows the two-way ANOVA p-values for the effects of genotype and disease stage, indicating that 2-oxoglutarate activity in hSOD1^G93A mice declines with progression of disease. The star denotes significance in the Bonferroni post test (* p<0.05).</p

Gene names, symbol, forward and reverse primer sequences and of primers used for the gene expression studies of metabolic genes.

<p>Gene names, symbol, forward and reverse primer sequences and of primers used for the gene expression studies of metabolic genes.</p

Triheptanoin preserves motor neurons.

<p>Starting at P35, female wild-type and hSOD1^G93A mice were either treated with triheptanoin (TRIH) or control (CON) diet until P70. (A) Stereologically counted motor neuron numbers in the L4-L5 segments of these 70 day old mice (n = 7–10) revealed a 38% loss of motor neurons in control-fed hSOD1^G93A mice. Triheptanoin provided a 33% protection against motor neuron loss. Two way ANOVA p<0.0001 for genotype, p = 0.0126 for treatment, the stars indicate results from a Bonferroni multiple comparisons post hoc tests if significant (**** p<0.0001, * p<0.05) (B-D) Representative thionine stained spinal cord sections from an untreated wild-type mouse (B) and untreated (C) and triheptanoin-treated (D) hSOD1^G93A mice with arrows pointing to the motor neurons counted. Scale bar 100 μm.</p

Relative expression of the anaplerotic genes, pyruvate carboxylase <i>Pcx</i> and glutamic pyruvic transferases <i>Gpt1</i> and <i>2</i>.

<p>Starting at P35, female wild-type and hSOD1^G93A mice were either treated with triheptanoin (TRIH) or control (CON) diet until they were sacrificed. Gene expresssion is compared in the gastrocnemius muscle of 10 and 25 week old triheptanoin treated vs. untreated wild-type and hSOD1^G93A mice relative to housekeeping genes. N-numbers of each group used in all graphs are indicated in the top bar graphs. The insets above each graph show the p-values for the effects of genotype in two-way ANOVAs, while the effect of diet was p>0.05 for each bar graph. When significant, the results of Bonferroni post tests are shown by a star (* p<0.05), indicating that triheptanoin treatment prevented the decrease of expression in <i>Gpt2</i> mRNA.</p

Triheptanoin treatment increased levels of plasma β-hydroxybutyrate.

<p>Plasma β-hydroxybutyrate levels (mM) in 70 days old wild-type and hSOD1^G93A mice fed with either triheptanoin (TRIH) or control (CON) diet from day 35 to 70 (One-way ANOVA p = 0.042; Fisher’s LSD post test: *p<0.05, ***p<0.001, n = 10–12).</p

Gene expression of enzymes involved in the propionyl-CoA carboxylase pathway.

<p>Starting at P35, female wild-type and hSOD1^G93A mice were treated with triheptanoin (TRIH) or control (CON) diet until they were sacrificed. Relative expression of the α (<i>Pcca</i>) and β (<i>Pccb</i>) subunits of propionyl-CoA carboxylase and methylmalonyl mutase (<i>Mut</i>). Expresssion is compared in the gastrocnemius muscle of 10 and 25 week old wild-type and hSOD1^G93A mice untreated or treated with triheptanoin relative to housekeeping genes. N-numbers used for each group throughout the experiments are indicated in the top bar graphs. The insets above each graph show the p-values for the effects of genotype in two-way ANOVAs, while the effect of diet was p>0.05 for each bar graph. When significant, the results of Bonferroni post tests are indicated by a star (* p<0.05), indicating that triheptanoin treatment in hSOD1^G93A mice protected against lowered expression of <i>Pccb</i> and <i>Mut</i> mRNA.</p

<i>Gapdh</i>, <i>Pdha</i>, <i>Ogdh</i> and <i>Sdha</i> mRNA expression in gastrocnemius muscle after triheptanoin treatment.

<p>Starting at P35, female wild-type and hSOD1^G93A mice were treated with triheptanoin (TRIH) or control (CON) diet until they were sacrificed. Quantitative real time PCR analysis of <i>Gapdh</i>, <i>Pdha1</i>, <i>Ogdh</i> and <i>Sdha</i> of the gastrocnemius muscle of 10 and 25 week old wild-type and hSOD1^G93A mice untreated or treated with triheptanoin relative to house keeping genes. N-numbers of each group used in all graphs are indicated in the top bar graphs. The insets above each graph show the p-values for the effects of genotype in two-way ANOVAs, while the effect of diet was p>0.05 for each bar graph. When significant, the results of Bonferroni post tests are shown by stars (* p<0.05, ** p<0.01), showing that decreases of mRNA levels of several enzymes in hSOD1^G93A mice were not apparent with triheptanoin treatment.</p

Triheptanoin treatment delays the loss of hind limb grip strength in hSOD1^G93A mice.

<p>Starting at P35, female wild-type and hSOD1^G93A mice were treated with triheptanoin (TRIH) or control (CON) diet until they were sacrificed. (A) No differences in grip strength was observed between triheptanoin (green open triangles, n = 15) and control treated wild-type mice (black filled squares, n = 12). (B) The grip strength over time differed in triheptanoin treated (red crosses, n = 8) vs. untreated (blue empty circles, n = 5) hSOD1^G93A mice (p = 0.04, two way ANOVA), with treated mice having higher grip strength at 18 and 19.5 weeks (p<0.05 Bonferroni post-hoc test). (C) The onset of hind limb grip strength loss was delayed by 2.8 weeks in triheptanoin treated hSOD1^G93A mice when compared to untreated hSOD1^G93A mice (p = 0.002, t-test). (D) Overall hind limb grip strength shown as the area under the curve over time was increased in triheptanoin treated hSOD1^G93A mice compared to control treated hSOD1^G93A mice (p = 0.02, t-test). (E) The onset of balance loss in triheptanoin treated hSOD1^G93A mice was significantly delayed by 13 days (p = 0.0016, t-test). (F) Body weights over time were significantly different between triheptanoin treated vs. untreated wild-type mice and treated vs. untreated hSOD1^G93A mice. (G) The onset of body weight loss in triheptanoin treated vs. untreated hSOD1^G93A mice (n = 5) was delayed (p = 0.007, t-test). * p<0.05, ** p<0.01. The onset of body weight loss was defined as the day where a loss of more than 10% in an individual mouse occurred relative to the mean body weight from week 12 to 17 was observed. Also all subsequent three body weight measurements were ≤ 90% of the original mean weight.</p