ALSUntangled #74: Withania Somnifera (Ashwagandha).

ALSUntangled reviews alternative and off-label treatments on behalf of people with ALS (PALS) who ask about them. Here, we review withania somnifera (WS) commonly known as ashwagandha or winter cherry. WS has plausible mechanisms for slowing ALS progression because of its effects on inflammation, oxidative stress, autophagy, mitochondrial function, and apoptosis. Preclinical trials demonstrate that WS slows disease progression in multiple different animal models of ALS. Of the five individuals we found who described using WS for their ALS, two individuals reported moderate benefit while none reported experiencing any significant side effects. There is currently one clinical trial using WS to treat PALS; the results are not yet published. There are no serious side effects associated with WS and the associated cost of this treatment is low. Based on the above information, WS appears to us to be a good candidate for future ALS trials.

ALSUntangled #70: caffeine.

ALSUntangled reviews alternative and off-label treatments for people living with amyotrophic lateral sclerosis (PALS). Here, we review caffeine which has plausible mechanisms for slowing ALS progression. However, pre-clinical studies are contradictory, and a large case series showed no relationship between caffeine intake and ALS progression rate. While low doses of caffeine are safe and inexpensive, higher doses can cause serious side effects. At this time, we cannot endorse caffeine as a treatment to slow ALS progression.

Early deficits in GABA inhibition parallels an increase in L-type Ca2+ currents in the jaw motor neurons of SOD1G93A mouse model for ALS.

Amyotrophic Lateral Sclerosis (ALS) involves protracted pre-symptomatic periods of abnormal motor neuron (MN) excitability occurring in parallel with central and peripheral synaptic perturbations. Focusing on inhibitory control of MNs, we first compared longitudinal changes in pre-synaptic terminal proteins for GABA and glycine neurotransmitters around the soma of retrogradely identified trigeminal jaw closer (JC) MNs and ChAT-labeled midbrain extraocular (EO) MNs in the SOD1G93A mouse model for ALS. Fluorescence immunocytochemistry and confocal imaging were used to quantify GAD67 and GlyT2 synaptic bouton density (SBD) around MN soma at pre-symptomatic ages ∼P12 (postnatal), ∼P50 (adult) and near disease end-stage (∼P135) in SOD1G93A mice and age-matched wild-type (WT) controls. We noted reduced GAD67 innervation in the SOD1G93A trigeminal jaw closer MNs around P12, relative to age-matched WT and no significant difference around P50 and P135. In contrast, both GAD67 and GlyT2 innervation were elevated in the SOD1G93A EO MNs at the pre-symptomatic time points. Considering trigeminal MNs are vulnerable in ALS while EO MNs are spared, we suggest that upregulation of inhibition in the latter might be compensatory. Notable contrast also existed in the innate co-expression patterns of GAD67 and GlyT2 with higher mutual information (co-dependency) in EO MNs compared to JC in both SOD1G93A and WT mice, especially at adult stages (P50 and P135). Around P12 when GAD67 terminals expression was low in the mutant, we further tested for persistent GABA inhibition in those MNs using in vitro patch-clamp electrophysiology. Our results show that SOD1G93A JC MNs have reduced persistent GABA inhibition, relative to WT. Pharmacological blocking of an underlying tonically active GABA conductance using the GABA-α5 subunit inverse agonist, L-655-708, disinhibited WT JC MNs and lowered their recruitment threshold, suggesting its role in the control of intrinsic MN excitability. Quantitative RT-PCR in laser dissected JC MNs further supported a reduction in GABA-α5 subunit mRNA expression in the mutant. In light of our previous report that JC MNs forming putative fast motor units have lower input threshold in the SOD1G93A mice, we suggest that our present result on reduced GABA-α5 tonic inhibition provides for a mechanism contributing to such imbalance. In parallel with reduced GABA inhibition, we noted an increase in voltage-gated L-type Ca2+ currents in the mutant JC MNs around P12. Together these results support that, early modifications in intrinsic properties of vulnerable MNs could be an adaptive response to counter synaptic deficits.

Therapeutic Approaches Targeting Protein Aggregation in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease that targets motor neurons (MNs) in the brain and spinal cord. It leads to gradual loss of motor signals to muscles leading to atrophy and weakness. Most patients do not survive for more than 3-5 years after disease onset. Current ALS treatments provide only a small delay of disease progression. Therefore, it is of utmost importance to explore new therapeutic approaches. One of the major hindrances in achieving this goal is poor understanding of causes of the disease. ALS has complex pathophysiological mechanisms in its genetic and sporadic forms. Protein aggregates are a common hallmark of ALS regardless of cause making protein pathways attractive therapeutic targets in ALS. Here, we provide an overview of compounds in different stages of pharmacological development and their protein pathway targets.

The molecular tweezer CLR01 inhibits aberrant superoxide dismutase 1 (SOD1) self-assembly in vitro and in the G93A-SOD1 mouse model of ALS.

Mutations in superoxide dismutase 1 (SOD1) cause 15-20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino acid substitutions destabilize SOD1's protein structure, leading to its self-assembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01-a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins-as a potential therapeutic agent for ALS. Using recombinant WT and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated mice, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other less aggressive ALS models may be needed to determine the therapeutic potential of this approach.