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ALS is a progressive neurodegenerative disease that affects neurons that control voluntary muscle movements. Environmental toxins such as pesticides and fungicides have been found to be potential causal factors for ALS as they may lead to the formation of toxic TDP-43 aggregation, a hallmark of the disease. Reactive oxygen species (ROS) lead to the formation of these toxic aggregates causing cell death. This study examined whether four pesticides: TS1, TS3, TS21, and TS31, induce toxic TDP-43 aggregation and whether oxidative stress is involved in response to the pesticides. Two experiments were conducted with primary cortical neurons that were isolated from wildtype mice embryos. Neurons were co-exposed to a sublethal dose exposure of the pesticides and reactive oxygen species inhibitors (cotreatments). Results showed that TS1 and TS3 caused TDP-43 toxic aggregation. Additionally, cotreatments that chelated zinc and copper had various degrees of recovery depending on the drug and cotreatment dosage. TS21 and TS31 did not cause toxic TDP-43 aggregation unless cotreated with ZnCl, which increased the intracellular levels of zinc. Further studies must be done with these toxins to determine how they lead to ALS pathology. Key Words: ALS, TDP-43 aggregation, reactive oxygen species, zinc, pesticides

Additional file 4: Figure S3. CA analysis in a refined AD cohort illustrates a correlation with cognition and APOE status. A Scatter plot of CA/mm2 vs AD Neuropathology score, ranked 0–3 (0 = none, 1 = mild, 2 = moderate, 3 = severe) for 112 patients, 12 outliers excluded. Spearman r = -−0.2728, **p = 0.0046. B Plot of CA/mm2 of cognitively normal and demented patients at Braak Stages II, III, and IV. (112 patients, 12 outliers excluded). * p = 0.033 (Braak II), 0.021 (Braak III). C Plot of CA/mm2 in E3/E3 vs E3/E4 patients at Braak Stages I, II, III, IV, and V. (112 patients, 12 outliers excluded). *p = 0.0198. D Plot of CA/mm2 in E3/E3 vs E3/E4 and E4/E4 patients at Braak stages IV, V and VI. (112 patients, 12 outliers excluded).

Additional file 1: Figure S1. CA analysis of AD cases spanning Braak stages I–VI. A Representative brightfield images of full dentate gyrus architecture in patient tissue samples from Braak I–VI. Inset (dashed box) indicates sample region used in Fig. 2A. Scale bars = 900 µm (I, IV), 500 (III) 400 µm (V), 600 µm (II, VI).

Additional file 2: Figure S1. B CA/mm2 distribution plot for all 124 patients included in this study showing a skewed distribution of patient CA/mm2. C Logarithmic distribution plot of CA/mm2 for all 124 included in this study. D Box plot of CA/mm2 vs Braak Stage using all 124 patients included in this study. E Logarithmic box plot of CA/mm2 vs Braak Stage (I–VI) displaying all 124 patients included in this study.

Additional file 3: Figure S2. CA analysis of a refined AD cohort lacking outliers. Scatter plot of refined patient cohort, CA Density vs Braak Stage I, II, III, IV, V, and VI. (112 patients in black, 12 outliers identified by Grubb’s test, Q = 0.05, excluded patients in red). Statistically identified outliers are depicted in the table. Pathological and demographic information for outliers excluded from analysis, sorted by Braak stage. From left to right: Alzheimer’s neuropathological score, age, APOE allele, cognitive status, post-mortem interval, sex, dentate gyrus area, CA numerical count, and CA/mm2.

Amyotrophic Lateral Sclerosis (ALS) is a progressing motor neuron disease that affects neurons responsible for voluntary muscle movements such as walking, talking, and breathing. While therapies are developed to treat ALS symptoms, there is no cure for the disease. Toxic environmental chemicals such as pesticides have been found to increase the likelihood of ALS by causing the toxic aggregation of TDP-43, the hallmark protein of the disease. TDP-43 is typically found in the nucleus of neurons. However, in ALS, it delocalizes to the cytoplasm and forms toxic aggregates. Oxidative stress is another factor known to induce TDP-43 aggregation. This study examines whether two pesticides, TS1 and TS3, cause toxic TDP aggregation. Oxidative stress is also studied to see whether it is involved in response to pesticides. Results of this study show that TS1 and TS3 induce toxic TDP-43 aggregation. It is also observed that oxidative stress may play a role in toxic TDP-43 aggregation formation. Further examination of these pesticides and the effects of oxidative stress can lead to novel druggable targets for ALS therapy.

Additional file 5: Figure S4. The detection of human CA in CSF requires primary IgM antibodies. A Shown is a secondary only control stain for immunofluorescent detection of CA in human patient CSF. No CA are detected in the absence of primary IgM antibody, though we note the detection of some non-specific debris. AlexaFluor anti-Ms IgG1 (red) and Anti-Ms IgM (green, brightness increased for visualization). Scale bar = 100 µm. Bottom row frames are insets from top row frames within the dashed white compartment.