AMPK Signalling and Defective Energy Metabolism in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is caused by selective loss of upper and lower motor neurons by complex mechanisms that are incompletely understood. Motor neurons are large, highly polarised and excitable cells with unusually high energetic demands to maintain resting membrane potential and propagate action potentials. This leads to higher ATP consumption and mitochondrial metabolism in motor neurons relative to other cells. Here, we review increasing evidence that defective energy metabolism and homeostasis contributes to selective vulnerability and degeneration of motor neurons in ALS. Firstly, we provide a brief overview of major energetic pathways in the CNS, including glycolysis, oxidative phosphorylation and the AMP-activated protein kinase (AMPK) signalling pathway, while highlighting critical metabolic interactions between neurons and astrocytes. Next, we review evidence from ALS patients and transgenic mutant SOD1 mice for weight loss, hypermetabolism, hyperlipidemia and mitochondrial dysfunction in disease onset and progression. Genetic and therapeutic modifiers of energy metabolism in mutant SOD1 mice will also be summarised. We also present evidence that additional ALS-linked proteins, TDP-43 and FUS, lead to energy disruption and mitochondrial defects in motor neurons. Lastly, we review emerging evidence including our own that dysregulation of the AMPK signalling cascade in motor neurons is an early and common event in ALS pathogenesis. We suggest that an imbalance in energy metabolism should be considered an important factor in both progression and potential treatment of ALS.     

Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes

Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.     

A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites

The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P₂ substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5–7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P₂ position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P₂ Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.     

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1^G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1^G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1^G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS.