Nandrolone Abuse Prior to Head Trauma Mitigates Endoplasmic Reticulum Stress, Mitochondrial Bioenergetic Deficits, and Markers of Neurodegeneration

Abstract

The abuse of synthetic steroids, such as nandrolone decanoate (ND), is often associated with violent behavior, increasing the risk of traumatic brain injury (TBI). After a TBI, proteins like APP, β-amyloid peptide-42 (Aβ42), and phosphorylated tau (pTau) accumulate and trigger endoplasmic reticulum (ER) stress associated with an unfolded protein response (UPR). The involvement of mitochondrial bioenergetics in this context remains unexplored. We interrogate whether the abuse of ND before TBI alters the responses of ER stress and mitochondrial bioenergetics in connection with neurodegeneration and memory processing in mice. Male CF1 adult mice were administered ND (15 mg/kg) or vehicle (VEH) s.c. for 19 days, coinciding with the peak day of aggressive behavior, and then underwent cortical controlled impact (CCI) or sham surgery. Spatial memory was assessed through the Morris water maze task (MWM) post-TBI. In synaptosome preparations, i) we challenged mitochondrial complexes (I, II, and V) in a respirometry assay, employing metabolic substrates, an uncoupler, and inhibitors; and ii) assessed molecular biomarkers through Western blot. TBI significantly increased APP, Aβ42, and pTau^Ser396 levels, along with ER-stress proteins, GRP78, ATF6, and CHOP, implying it primed apoptotic signaling. Concurrently, TBI reduced mitochondrial Ca²⁺ efflux in exchange with Na⁺, disturbed the formation/dissipation of membrane potential, increased H₂O₂ production, decreased biogenesis (PGC-1⍺ and TOM20), and ATP biosynthesis coupled with oxygen consumption. Unexpectedly, ND abuse before TBI attenuated the elevations in APP, Aβ42, and pTau^Ser396, accompanied by a decrease in GRP78, ATF6, and CHOP levels, and partial normalization of mitochondrial-related endpoints. A principal component analysis revealed a key hierarchical signature featuring mitochondrial Ca²⁺ efflux, CHOP, GRP78, TOM20, H₂O₂, and bioenergetic efficiency as a unique variable (PC1) able to explain the memory deficits caused by TBI, as well as the preservation of memory fitness induced by prior ND abuse.

Keywords: Abuse; Nandrolone; Traumatic brain injury.

Fig. 1.. Experimental timeline.

Sixty days old CF1 albino mice were randomly allocated to 25 days experimental protocol. Mice received for 19 days nandrolone decanoate (ND) or oil-vehicle. Afterwards mice were randomly allocated into the groups that underwent either a sham operation or a CCI to induce TBI resulting in VEH/SHAM, ND/SHAM, VEH/CCI, and ND/CCI (n = 5–6 mice/group). On day 21^st mice were submitted to open field test, and from day 22 to 25^th to Morris water maze training (WM training) and test (WMT). After the Morris water maze task mice were euthanized, and the brain was dissected for biochemical analysis. Abbreviations: D, day; VEH, vehicle; ND, nandrolone decanoate; CCI, controlled cortical impact; WM, Morris water maze; WMT, Morris water maze test; EU, euthanasia; BA, biochemical assays.

Figure 2.. Brain accumulated proteins.

The immunocontent of proteins whose accumulation in the brain is associated with neurodegenerative mechanisms was estimated using Western blot analysis with synaptosomes preparation from whole-brain mice. A traumatic brain injury (CCI) led to a significant increase in the levels of (A) Protein precursor amyloid (APP); (B) Amyloid-β peptide 42 (Aβ-42); and (C) Phosphorylated Tau at serine 396 (p-Tau^ser396) and total Tau protein (Tau) ratio. Statistical differences was notice in the following comparisons: (A) VEH/CCI vs ND/SHAM; (B) VEH/CCI vs VEH/SHAM; (C) VEH/CCI vs VEH/SHAM; and VEH/CCI vs ND/SHAM. Nandrolone prior to TBI sustained APP and Aβ-42 at the level of VEH/SHAM (n=5–6 mice per group). * Indicates statistical significance at p<0.05 for all comparisons. Data is presented as Mean ± S.D. Abbreviations: VEH, vehicle; ND, nandrolone decanoate; CCI, controlled cortical impact.

Figure 3.. Endoplasmic reticulum (ER) stress.

The immunocontent of proteins that serve as biomarkers of ER stress was estimated using Western blot analysis with synaptosomes preparation from whole-brain mice. A trauma to the brain (CCI) led to a significant upregulation in the abundance of (A) 78-kDa glucose-regulated protein (GRP-78); (B) Activating transcription factor 6 (ATF-6); and (C) C/EBP homologous protein (CHOP). Nandrolone prior to TBI sustained GRP-78, ATF-6, and CHOP at the level of VEH/SHAM (n= 5 mice per group). * Indicates statistical significance at p<0.05 for all comparisons. Data are shown as mean ± S.D. Abbreviations: VEH, vehicle; ND, nandrolone decanoate; CCI, controlled cortical impact.

Figure 4.. Mitochondrial plasticity biomarkers.

The immunocontent of proteins related to mitochondrial biogenesis and capability to import cytosolic proteins to the matrix was assessed using Western blot analysis with synaptosomes preparation from whole-brain mice. A traumatic brain injury causes downregulation in the abundance of (A) Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α); (B) Translocase of outer membrane 20 (TOM 20). Nandrolone prior to CCI sustained PGC1-α and TOM20 at the level of VEH/SHAM (n= 5 mice per group). * Indicates statistical significance at p<0.05 for all comparisons. Data are shown as mean ± S.D. Abbreviations: VEH, vehicle; ND, nandrolone decanoate; CCI, controlled cortical impact.

Figure 5.. Panel of mitochondrial-related bioenergetic endpoints.

Mitochondrial function was assessed in synaptosomes from whole-brain mice. (A) Mitochondria energized with metabolic substrates for the complexes I, II and V (pyruvate, malate, glutamate, succinate, and ADP; PMGSA), were challenged regarding Ca²⁺ influx (swelling after Ca²⁺ addition) and Ca²⁺ efflux (shrinkage after Na⁺ addition) capacity. VEH/SHAM showed significantly less swelling relative to other groups. Calcium efflux was impaired in VEH/CCI relative to other groups, and ND prior to CCI partially sustained the capacity of mitochondria to extrude calcium (* p<0.05 for all comparisons). (B) Formation and dissipation of mitochondrial membrane potential (Δψm) in relation to mitochondrial H₂O₂ production. VEH/CCI group showed a lower Δψm concurrent with increased H₂O₂ production when compared to VEH/SHAM after the addition of: i) substrates to complexes I and II (CI+CII), and ii) Complex V (CI+CII (P)); iii) an inhibitor of Complex V (Lomy); and iv) the uncoupler UFCCP (*p<0.05). ND/CCI group displayed different values of Δψm and H₂O₂ (CI+CII and CI+CII(P)) relative to VEH/SHAM (#p<0.05). Relative to VEH/CCI, ND/CCI alleviated the disruption in Δψm, leading to a reduced H₂O₂ production particularly in Lomy (# p<0.05). In the UFCCP state, ND/CCI sustained the H₂O₂ production at the level of VEH/SHAM. The bars below the dashed lines represent Δψm formation and bars above dashed lines represent Δψm dissipation. The columns above dashed lines represent H₂O₂ production. (C) Oxygen consumption rate in real-time respirometry after challenging synaptosomes with metabolic substrates to CI (PMG), CII (S), CV (ADP), an inhibitor of CV(oligomycin); and an uncoupler (UFCCP). In the basal state, ND/SHAM showed significant increase in oxygen consumption relative to VEH/SHAM and VEH/CCI. Further, VEH/CCI displayed decreased respiratory capacity in CI+CII(P) and uncoupling (UFCCP) states relative to VEH/SHAM (*p<0.05). In UFCCP state, ND/CCI was different from VEH/SHAM (# p<0.05). The following parameters (D) Proton leak; (E) Electron transfer efficiency; and (F) Biochemical coupling efficiency, were significantly impaired in VEH/CCI relative to VEH/SHAM, ND/SHAM, and ND/CCI (*p<0.05). Prior nandrolone administration attenuated the impairment in mitochondrial bioenergetics caused by CCI. (n=5–6 per group). Data are shown as mean ± S.D. Abbreviations: VEH, vehicle; ND, nandrolone decanoate; CCI, controlled cortical impact.

Figure 6.. System biology approach of molecular and functional endpoints.

(A) Correlogram of ER stress biomarkers and mitochondrial variables. The bivariate correlation analysis confirms a significant association between ER stress and mitochondrial endpoints, suggesting that they may exert varying degrees of positive and/or negative functional influence on each other within the range of −1 to +1. (B) Principal component analysis (PCs). The molecular and biochemical variables were grouped in 9 PCAs being that PC1 seemed to explain 87 % of the variances found in the results. (C) Variable contribution to PC1. The variables that contribute to the PC1 were plotted according to the higher to lower magnitude contribution. Six variables that cross beyond the dashed red line presented a statistical significant contribution to the PC1. (D) Group profiling of PC1. Considering the six significant variables, the PC1 values can effectively provide a discrimination among all groups (*p<0.05). Abbreviations: BCE, biochemical coupling efficiency; ETS, electron transfer system capacity; Δψm P, variation of mitochondrial membrane potential (ADP); Δψm Lomy variation of mitochondrial membrane potential (olygomycin); H₂0₂ P, mitochondrial hydrogen peroxide production (ADP); H₂0₂ Lomy, mitochondrial hydrogen peroxide production (oligomycin); APP, Amyloid precursor protein; PGC1-α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; TOM20, Translocase of outer membrane 20; GRP-78, 78-kDa glucose-regulated protein; ATF-6, Activating transcription factor 6; and CHOP, C/EBP homologous protein.

Figure 7.. Spatial memory task.

Spatial memory was assessed using the Morris water maze. (A) During the 3-day learning phase, mice from all groups successfully learned to locate a submerged hidden platform (*p<0.05). On the third day of training, mice in the VEH/CCI group spent more time searching for the platform compared to the other groups (^#p<0.05). (B) In the test phase, mice from VEH/CCI group spent less time in the quadrant where the hidden platform was previously located (*p<0.05). (C) Average speed did not differ between groups. (D) Correlation between PC1 and Time (seconds) in the target quadrant (r= −0.944, p=0.0001). The dot colors indicate treatment groups: Red dots for VEH/SHAM; grey dots for ND/SHAM; green dots for VEH/CCI, and the black dots for ND/CCI. (E) A representative image is provided for one mouse from each group during the test trial. Abbreviations: VEH, vehicle; ND, nandrolone decanoate; CCI, controlled cortical impact; MWT, Morris water maze test