Fig 2: Overview of the potential physiological mechanisms during the early stages of disuse in humans. (1) After 5 days of disuse, the release of light neurofilaments suggests axonal damage, which may progressively cause NMJ instability (2) through the cleavage of agrin and the expression of NCAM, which show significance at 10 days of disuse. NMJ instability is associated with (3) reduced RtkB neurotrophin receptors for BDNF and NT-4, (4) which may be responsible for the marked sarcolemmal hemichannels’ upregulation after 10 days of disuse. In turn, hemichannels may start to release ATP with paracrine properties, (5) acting on purinergic receptors, (6) resulting in the transcription of inflammatory transcripts and (7) in muscle atrophy, with the progression of disuse. Indeed, (8) sarcolemmal hemichannels upregulation may cause ionic disbalance along the sarcolemma, resulting in unstable membrane potential at rest and a reduced capacity to generate efficacious excitation-contraction coupling cascades, resulting in reduced muscle strength.

Fig 3: Effect of Hap-Car on osteogenic markers and trophic factors. hFOB1.19 cells were stimulated with Hap (0.05 mg/mL), Hap-Car10:1, Hap-Car2:1, and Hap-Car1:1 with or without 50 μM BCS for 4dd. Densitometric analysis (A) and representative Western blot image (B) of BMP-2 expression levels. The expression level of BMP-2 is reported as % of control of ratio over Actin. (C) The quantitative analysis of fluorescence for BMP-2 expression was analyzed using ImageJ Software1.53 and is expressed as fluorescence intensity normalized with the number of cells per photo. (D) Representative images of hFOB1.19 cells acquired by fluorescence microscopy after 4dd treatment. The cells were incubated with anti-BMP-2 antibody (green) and nuclear dye Hoechst33348 (blue). Scale bar 65.8 μm, magnification 40×. The extracellular release of BMP-2 (E), osteocalcin (F), BDNF (G), and VEGF (H) is reported as a ratio over the cell number calculated by Incucyte software and reported as % of control. Data are expressed as mean ± SD. Statistical significance is indicated as *p < 0.05; ** p < 0.01, and *** p < 0.001.

Fig 4: Photocleavable nanoparticle (PCN) fabrication. (a) Schematic illustration of the self-assembly of polyethylene glycol octamethylene diamine–ethanolamine − leucomethylene blue (PEGOD-EA-LMB) polymer and encapsulation of brain-derived neurotropic factor (BDNF) in aqueous–-organic–aqueous solutions to form approximately 50 nm PCNs as observed by (b) transmission electron microscopy with hydrophobic LMB core and hydrophilic PEGOD tail. Loading amounts higher than 5 µg with either (c) bovine serum albumin or (d) BDNF did not increase encapsulation efficiency. The statistical significance of the data was analyzed using one-way ANOVA with Tukey’s multiple comparisons test for each group (n = 6)

Fig 5: Impaired hippocampal neurogenesis in the SCA1 mice. (a) Representative images of hippocampal sections stained for NeuN. Scale bars (a,b) = 300 µm. (b) Representative images of the DG double stained for DCX and PSA-NCAM (with DAPI). (c) NeuN immunofluorescence intensity in the CA1 (left) and CA2/3 (middle-left) pyramidal layers and the density of NeuN+ neurons (per 1,000 µm2) in the same hippocampal regions (right). (d) Density of DCX+PSA-NCAM+ neurons per 100 µm of the DG subgranular zone. (e) Density of DCX+ dendrites crossing the line on the border of the DG-G and DG-ML (M/G; left), inner part of the DG-ML (M-in; middle) and outer part of the DG-ML (M-out; right), per 100 µm. (f) PSA-NCAM immunofluorescence in the DG polymorph layer (left; DG-P), DG-ML (middle-left), CA4 pyramidal (middle-right; CA4-P) and CA1 stratum lacunosum-moleculare (right; CA1) hippocampal layers. N (c-f) = 5 animals per group (with 4 sections per animal). Each point = 1 section. The statistical significances were based on the permutational linear mixed-effect models (See Suppl. Tables S25 and S26 for the detailed results) using 4 replicates per animal and with the animal identity representing the random-effect factor. (g) Hippocampal BDNF level (pg/mg of proteins). N = 6 animals per group. P = 0.046 (permutational t-test). Each point = 1 animal. Box-whisker plots (c–g) indicating the inter-quartile (IQ) intervals (box), 1.5*IQ range (whiskers) and medians (middle line). *P < 0.05, **P < 0.01, ***P < 0.001. n.s. = not significant.