Fig 2: Innervation of mineralized cell-laden collagen. a Timeline for the generation of innervated and mineralized collagen constructs. Human SH-SY5Y neuroblastoma cells were co-encapsulated with hMSCs (4:1) in non-mineralized and mineralized hydrogels, after 14 days of differentiation. For the first 7 days, the cells were treated in DMEM/F-12 basal medium supplemented with 1% FBS and 10 µM all-trans retinoic acid (RA), followed by an additional 7 days of culture in Neurobasal-A medium containing 1% (v/v) l-glutamine, 1× B-27 supplement, 50 ng/mL human BDNF, and 10 µM RA. Subsequently, matrix mineralization was triggered by switching the neurobasal medium for the mineralizing medium for another 3 days. The fully differentiated neuronal cells within the mineralized constructs were confirmed by immunostaining with antibodies against Neuron-specific enolase (NSE) and Neurofilament light (NEFL). Cell nuclei were counterstained with DAPI (blue) and cytoskeletal actin was stained with Alexa Fluor 488 phalloidin (green). b, c Representative immunofluorescence images showing the expression of NSE and NEFL in non-mineralized and mineralized constructs. Both the neuronal differentiation markers had similar expression levels in mineralized and non-mineralized groups. Scale bar: 50 µm. d Imaris Filament Tracer module was used to quantify the morphological parameters of the differentiated SHSY-5Y cells in non-mineralized vs. mineralized constructs. Quantification of the number and length of neurites indicate no significant difference between non-mineralized and mineralized groups, whereas the number of branches and branch points was higher in the mineralized groups. Data are represented as Mean ± SD, *p < 0.05, Student’s t-test (N = 4). Source data are provided as a Source Data file

Fig 3: Decreased NF-L protein in LPS-treated HNG cells and in AD: ELISA and Western analysis. (A) results of sandwich ELISA analysis for NF-L protein in LPS-treated HNG cells using Abbexa (abx250460; Cambridge, United Kingdom) and/or LifeSpan BioSciences (LSBio; LS-F6701; Seattle WA, United States); the 68 kDa NF-L species is a particularly abundant intermediate filament protein, however in the presence of LPS the abundance of NF-L protein was found to be reduced to about 0.3-fold of control; a dashed horizontal line at 100 is included for ease of comparison; N = 3 to 5 experiments per determination; *p < 0.01 (ANOVA); (B) Western analysis of total NF-L protein (MW ~68 kDa) in control (pool of five controls and five AD temporal lobe neocortex Brodman A22) and total NF-L protein in control and LPS-treated HNG cells (at 2 weeks of culture; see Figure 4A); ß-actin protein (MW ~42 kDa) was used as an internal control marker in the same sample for each determination; (C) Western blots were quantified in bar graph format of decreased NF-L protein abundance in AD neocortex versus age-matched controls and in LPS-treated HNG cells versus age-matched controls; a dashed horizontal line at 1.0 is included for ease of comparison; the results of decreased NF-L expression for AD over control or LPS-treated HNG cells over control are highly significant; N = 3 to 5 experiments; *p < 0.01 (ANOVA).

Fig 4: Microbiome-derived LPS-mediated impairment of NF-L expression may contribute to atrophy of neurons and cytoskeletal disorganization that is characteristic of sporadic AD – the human GI-tract microbiome secretes a remarkably heterogeneous and complex mixture of neurotoxins including different varieties of lipooligosaccahrides (LOS), lipopolysaccharide (LPS), amyloids, small non-coding RNAs (sncRNAs) and exotoxins; recently several laboratories have provided evidence that these neurotoxins may transit GI-tract and blood-brain barriers and are present in the CNS and within aged or AD brain tissues; whether these microbiome-derived neurotoxins originate from the gastrointestinal (GI) tract microbiome, a possible brain microbiome or some dormant pathological microbiome is currently not well understood. Recent studies further suggest that the co-localization of pro-inflammatory LPS with AD-affected neuronal nuclei provides evidence that there may be a contribution of LPS to genotoxic events that support deficits in homeostatic gene expression that drive progressive AD-type change and provide support for pro-inflammatory neurodegeneration. This communication provides evidence that in both LPS-enveloped neuronal nuclei in AD neocortex and LPS-treated HNG cells in primary co-culture that there is a significant deficit in the expression of the neurofilament light-chain (NF-L), a neuron-specific cytoskeletal element known to be important in maintaining the shape and synaptic integrity of the neuron; see text and Figures 1–5 for additional details.