Alexandro Gianforcaro

Vitamin D3 Deficiency Differentially Affects Functional and Disease Outcomes in the G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by motor neuron death in the central nervous system. Vitamin D supplementation increases antioxidant activity, reduces inflammation and improves motor neuron survival. We have previously demonstrated that vitamin D3 supplementation at 10× the adequate intake improves functional outcomes in a mouse model of ALS. Objective To determine whether vitamin D deficiency influences functional and disease outcomes in a mouse model of ALS. Methods At age 25 d, 102 G93A mice (56 M, 46 F) were divided into two vitamin D3 groups: 1) adequate (AI; 1 IU D3/g feed) and 2) deficient (DEF; 0.025 IU D3/g feed). At age 113 d, tibialis anterior (TA), quadriceps (quads) and brain were harvested from 42 mice (22 M and 20 F), whereas the remaining 60 mice (34 M and 26 F) were followed to endpoint. Results During disease progression, DEF mice had 25% (P = 0.022) lower paw grip endurance AUC and 19% (P = 0.017) lower motor performance AUC vs. AI mice. Prior to disease onset (CS 2), DEF mice had 36% (P = 0.016) lower clinical score (CS) vs. AI mice. DEF mice reached CS 2 six days later vs. AI mice (P = 0.004), confirmed by a logrank test which revealed that DEF mice reached CS 2 at a 43% slower rate vs. AI mice (HR = 0.57; 95% CI: 0.38, 1.74; P = 0.002). Body weight-adjusted TA (AI: r = 0.662, P = 0.001; DEF: r = 0.622, P = 0.006) and quads (AI: r = 0.661, P = 0.001; DEF: r = 0.768; P<0.001) weights were strongly correlated with age at CS 2. Conclusion Vitamin D3 deficiency improves early disease severity and delays disease onset, but reduces performance in functional outcomes following disease onset, in the high-copy G93A mouse.

Vitamin D as a Potential Therapy in Amyotrophic Lateral Sclerosis

Vitamin D has been demonstrated to influence multiple aspects of amyotrophic lateral sclerosis (ALS) pathology. Both human and rodent central nervous systems express the vitamin D receptor (VDR) and/or its enzymatic machinery needed to fully activate the hormone. Clinical research suggests that vitamin D treatment can improve compromised human muscular ability and increase muscle size, supported by loss of motor function and muscle mass in animals following VDR knockout, as well as increased muscle protein synthesis and ATP production following vitamin D supplementation. Vitamin D has also been shown to reduce the expression of biomarkers associated with oxidative stress and inflammation in patients with multiple sclerosis, rheumatoid arthritis, congestive heart failure, Parkinsons disease and Alzheimers disease; diseases that share common pathophysiologies with ALS. Furthermore, vitamin D treatment greatly attenuates hypoxic brain damage in vivo and reduces neuronal lethality of glutamate insult in vitro; a hallmark trait of ALS glutamate excitotoxicity. We have recently shown that high‐dose vitamin D3 supplementation improved, whereas vitamin D3 restriction worsened, functional capacity in the G93A mouse model of ALS. In sum, evidence demonstrates that vitamin D, unlike the antiglutamatergic agent Riluzole, affects multiple aspects of ALS pathophysiology and could provide a greater cumulative effect.

Dietary vitamin D3 supplementation at 10× the adequate intake improves functional capacity in the G93A transgenic mouse model of ALS, a pilot study.

Vitamin D has antioxidant, anti‐inflammatory, and neuroprotective properties, and may mitigate amyotrophic lateral sclerosis (ALS) pathology.

Vitamin D3 at 50x AI Attenuates the Decline in Paw Grip Endurance, but Not Disease Outcomes, in the G93A Mouse Model of ALS, and Is Toxic in Females

Background We previously demonstrated that dietary vitamin D3 at 10x the adequate intake (AI) attenuates the decline in functional capacity in the G93A mouse model of ALS. We hypothesized that higher doses would elicit more robust changes in functional and disease outcomes. Objective To determine the effects of dietary vitamin D3 at 50xAI on functional outcomes (motor performance, paw grip endurance) and disease severity (clinical score), as well as disease onset, disease progression and lifespan in the transgenic G93A mouse model of ALS. Methods Starting at age 25 d, 100 G93A mice (55 M, 45 F) were provided ad libitum with either an adequate (AI; 1 IU D3/g feed) or high (HiD; 50 IU D3/g feed) vitamin D3 diet. Results HiD females consumed 9% less food corrected for body weight vs. AI females (P = 0.010). HiD mice had a 12% greater paw grip endurance over time between age 60–141 d (P = 0.015), and a 37% greater score during disease progression (P = 0.042) vs. AI mice. Although HiD females had a non-significant 31% greater CS prior to disease onset vs. AI females, they exhibited a significant 20% greater paw grip endurance AUC (P = 0.020) when corrected for clinical score. Conclusion Dietary D3 supplementation at 50x the adequate intake attenuated the decline in paw grip endurance, but did not influence age at disease onset, hindlimb paralysis or endpoint in the transgenic G93A mouse model of ALS. Furthermore, females may have reached the threshold for vitamin D3 toxicity as evidence by reduced food intake and greater disease severity prior to disease onset.

Dietary Vitamin D3 Restriction Exacerbates Disease Pathophysiology in the Spinal Cord of the G93A Mouse Model of Amyotrophic Lateral Sclerosis

Background Dietary vitamin D3 (D3) restriction reduces paw grip endurance and motor performance in G93A mice, and increases inflammation and apoptosis in the quadríceps of females. ALS, a neuromuscular disease, causes progressive degeneration of motor neurons in the brain and spinal cord. Objective We analyzed the spinal cords of G93A mice following dietary D3 restriction at 2.5% the adequate intake (AI) for oxidative damage (4-HNE, 3-NY), antioxidant enzymes (SOD2, catalase, GPx1), inflammation (TNF-α, IL-6, IL-10), apoptosis (bax/bcl-2 ratio, cleaved/pro-caspase 3 ratio), neurotrophic factor (GDNF) and neuron count (ChAT, SMI-36/SMI-32 ratio). Methods Beginning at age 25 d, 42 G93A mice were provided food ad libitum with either adequate (AI;1 IU D3/g feed; 12 M, 11 F) or deficient (DEF; 0.025 IU D3/g feed; 10 M, 9 F) D3. At age 113 d, the spinal cords were analyzed for protein content. Differences were considered significant at P ≤ 0.10, since this was a pilot study. Results DEF mice had 16% higher 4-HNE (P = 0.056), 12% higher GPx1 (P = 0.057) and 23% higher Bax/Bcl2 ratio (P = 0.076) vs. AI. DEF females had 29% higher GPx1 (P = 0.001) and 22% higher IL-6 (P = 0.077) vs. AI females. DEF males had 23% higher 4-HNE (P = 0.066) and 18% lower SOD2 (P = 0.034) vs. AI males. DEF males had 27% lower SOD2 (P = 0.004), 17% lower GPx1 (P = 0.070), 29% lower IL-6 (P = 0.023) and 22% lower ChAT (P = 0.082) vs. DEF females. Conclusion D3 deficiency exacerbates disease pathophysiology in the spinal cord of G93A mice, the exact mechanisms are sex-specific. This is in accord with our previous results in the quadriceps, as well as functional and disease outcomes.