Fig 2: Suggested association between amino acids and DPN development. (a) Schematic showing direct relation between decreased metabolite NAA and decreased gene transcript NAT8L, created with BioRender. NAA is synthesized in the mitochondria from Asp by NAT8L. NAA is then transported to Schwann cells, where it is a precursor for myelin lipids. (b) Schematic showing suggested interplay between identified molecular changes, created with BioRender. Briefly, amino acid starvation can impair mitochondrial function as well as inhibit protein translation, resulting in ER stress. Impaired function of the mitochondria and ER can also lead to disrupted crosstalk between these organelles and thus disrupted phospholipid synthesis. Disrupted mitochondrial function can also impair NAA synthesis, impacting glial-neuronal structures. Changes to the extracellular matrix can be related to ER stress and amino acid uptake. Finally, both ER stress and amino acid depletion can induce hyperphosphorylation of eEF2, thus inhibiting protein translation. Known molecular relationships are marked with solid lines, while relationships which are suggested by literature, but not well studied are indicated with dashed arrows.

Fig 3: Effect of propofol anesthesia on Arc, eEF2, and ERK proteins levels in mice.Hippocampal Arc (1), ß-actin (2), phospho-eEF2 (3), eEF2 (4), phospho-ERK (5), and ERK (6) protein immunoblot relative band intensity (% of control) in C57BL/6J mice following exposure to propofol under hypothermic conditions. Hippocampal tissues from the propofol-anesthetized mice were obtained immediately at the end of anesthesia (Prop). ERK1 (44 kDa) and ERK2 (42 kDa) were quantified together. Two lanes from representative immunoblots for all the proteins are displayed for each condition. Dividing lines represent areas where lanes from the same blot were removed and the remaining lanes were spliced together. Original unaltered data can be found in Fig. S5. Data are expressed as mean ± SD, * and *** symbols denote a significant difference versus control with P < 0.05 and P < 0.001, respectively, n = 5 for Ctl and 4 for Prop.

Fig 4: eEF2K expression and activity in PD brain. a Quantitation of p-eEF2 (T56) IHC (3,3'-Diaminobenzidine-DAB staining) in postmortem hippocampus- Hip (CA1 and CA2 fields) and midbrain- MB (SN-substantia nigra, and PAG-peri-aqueductal gray matter) sections from 3 control and 6 PD cases (Additional file 1: Table S1; counts from at least 6 high power fields from each control or PD section; Mann–Whitney test, *p < 0.05, ***p < 0.005; error bars indicate Mean ± S.D.). b-d eEF2K mRNA expression in control and PD striatum (b), medial substantia nigra (b) and dorsal nucleus of vagus nerve (d). The following publicly available transcriptomic profile datasets were analyzed on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) platform: Striatum (b)- dataset GEO accession # GSE28894, Illumina human Ref-8 v2.0 expression beadchip platform, probe ID ILMN_1789171, controls n = 15 and PD n = 15; Medial substantia nigra (c)- dataset GEO accession # GSE8397, Affymetrix Human Genome U133B Array, probe ID 225546_at, controls n = 8 and PD n = 15; Dorsal nucleus of vagus (d)- dataset GEO accession # GSE43490, Agilent-014850 Whole Human Genome Microarray, probe ID A_24_P716162, controls n = 6 and PD n = 7. (Mann–Whitney test, *p < 0.05; error bars in 3b-d indicate Mean ± S.D.). e Relative eEF2K mRNA expression in human iPSCs derived cultured midbrain control (WT, n = 3) or A53T (n = 2) mutation carrying organoids (T-test, *p < 0.05; error bars indicate Mean ± S.D.)

Fig 5: Effect of isoflurane anesthesia on eEF2 and phospho-eEF2 protein levels in rats.Hippocampal phospho-eEF2 (1), and eEF2 (2) protein immunoblot relative band intensity (% of control) in Fischer 344 rats following exposure to isoflurane under hypothermic conditions (A) or normothermic conditions (B). Hippocampal tissues were sampled immediately at the end of anesthesia (IH: Isoflurane-Hypothermia, and IN: Isoflurane-Normothermia), or 24 h later (IH24h: Isoflurane-Hypothermia 24h, and IN24h: Isoflurane-Normothermia 24h). One lane from representative immunoblots for phospho-eEF2 and eEF2 are displayed for each condition. Dividing lines represent areas where lanes from the same blot were removed and the remaining lanes were spliced together. Original unaltered data from two batches of Fisher 344 rats can be found in Fig. S2. Data are expressed as mean ± SD, * and ** symbols denote a significant difference versus control with P < 0.05 and P < 0.01, respectively, n = 8 for all conditions.