Irina Vazquez-Villaseñor

Cellular senescence may affect the post-mitotic cells of the brain. We examined the expression of senescence markers, including p16, p21, γH2Ax and H3K9me3, in the frontal cortex of brain donations from the Cognitive Function and Ageing Study to assess their relationship to Alzheimer’s disease neuropathological change (ADNC) and dementia. p21, γH2Ax and H3K9me3 were expressed in pyramidal neurons and glia, whilst p16 was confined to glial cells. p21 and γH2Ax were correlated in neurons, and with p16 in glia. They did not increase with ADNC, tending to be higher at early Braak neurofibrillary tangle stages. Transcriptomic profiling of pyramidal neuron-enriched samples at low Braak stages showed that higher neuronal p21 expression was associated with altered pathways for neuronal function, neurodegeneration, protein homeostasis, mitochondrial dysfunction and synaptic signalling. In conclusion, the different expression profile of senescence markers in neurons and glia suggest possible differences in senescence-related mechanisms. Expression at lower ADNC stages suggests senescence may be important at earlier stages of Alzheimer’s pathogenesis, whilst transcriptomic changes suggest an impact on neuronal function. The lack of association of senescence markers with dementia status indicates that more work is needed to determine the value of senescence as a therapeutic target for dementia.

(1) Background: Systemic infection is associated with increased neuroinflammation and accelerated cognitive decline in AD patients. Activated neutrophils produce neutrophil-derived microvesicles (NMV), which are internalised by human brain microvascular endothelial cells and increase their permeability in vitro, suggesting that NMV play a role in blood–brain barrier (BBB) integrity during infection. The current study investigated whether microRNA content of NMV from AD patients is significantly different compared to healthy controls and could impact cerebrovascular integrity. (2) Methods: Neutrophils isolated from peripheral blood samples of five AD and five healthy control donors without systemic infection were stimulated to produce NMV. MicroRNAs isolated from NMV were analysed by RNA-Seq, and online bioinformatic tools were used to identify significantly differentially expressed microRNAs in the NMV. Target and pathway analyses were performed to predict the impact of the candidate microRNAs on vascular integrity. (3) Results: There was no significant difference in either the number of neutrophils (p = 0.309) or the number of NMV (p = 0.3434) isolated from AD donors compared to control. However, 158 microRNAs were significantly dysregulated in AD NMV compared to controls, some of which were associated with BBB dysfunction, including miR-210, miR-20b-5p and miR-126-5p. Pathway analysis revealed numerous significantly affected pathways involved in regulating vascular integrity, including the TGFβ and PDGFB pathways, as well as Hippo, IL-2 and DNA damage signalling. (4) Conclusions: NMV from AD patients contain miRNAs that may alter the integrity of the BBB and represent a novel neutrophil-mediated mechanism for BBB dysfunction in AD and the accelerated cognitive decline seen as a result of a systemic infection.

Oxidative DNA damage induces changes in the neuronal cell cycle and activates a DNA damage response to promote repair, but these processes may be altered under a chronic oxidative environment, leading to the accumulation of unrepaired DNA damage and continued activation of a DNA damage response. Failure to repair DNA damage can lead to apoptosis or senescence, which is characterized by a permanent cell-cycle arrest. Increased oxidative stress and accumulation of oxidative DNA damage are features of brain ageing and neurodegeneration but the effects of persistent DNA damage in neurons are not well-characterized. We developed a model of persistent oxidative DNA damage in immortalized post-mitotic neurons in vitro by exposing them to a sub-lethal concentration of hydrogen peroxide following a “double stress” protocol, and performed a detailed characterization of the neuronal transcriptome using microarray analysis. Persistent DNA damage significantly altered the expression of genes involved in cell cycle regulation, DNA damage response and repair mechanisms, and mitochondrial function, suggesting an active DDR response to replication stress and alterations in mitochondrial electron transport chain. qPCR and functional validation experiments confirmed hyperactivation of mitochondrial Complex I in response to persistent DNA damage. These changes in response to persistent oxidative DNA damage may lead to further oxidative stress, contributing to neuronal dysfunction and ultimately neurodegeneration.

Aims: Cellular senescence plays a role in organismal ageing and has been linked to persistent DNA damage in age‐related diseases. Brain senescence has been described in astrocytes and microglia, but it is less well understood in neurones. Evidence suggests that neurones activate a senescence‐like mechanism that could contribute to neurodegeneration. We aimed to determine whether a persistent DNA damage response (DDR) and senescence activation are features of motor neurone disease (amyotrophic lateral sclerosis, ALS/MND).Methods: We examined expression of senescence (p16 and p21) and DNA damage markers (8‐OHdG and γH2AX) in motor cortex (MCx), frontal association cortex (FACx) and occipital cortex (OCx) in post‐mortem tissue donated by patients with ALS/MND and controls.Results: Nuclear expression of p16 and p21 was detected in glial cells; double immunofluorescence for p16/p21 and glial fibrillary acidic protein (GFAP) suggested that some of these cells were GFAP⁺ astrocytes. p21 nuclear expression was also found in neurones. Higher levels of p16⁺ (glia, P = 0.028) and p21⁺ (glia, P = 0.003; neurones, P = 0.008) cells were found in the FACx of ALS/MND donors but not in the MCx or OCx. Expression of p16 and p21 did not correlate with 8‐OHdG or γH2AX.Conclusions: Expression of p16 and p21 in glia, mainly in astrocytes, suggests senescence induction in these cells; however, neuronal p21 expression might reflect a more general mechanism of age‐related cell cycle dysregulation. The significantly higher proportion of cells expressing either p16 or p21 in the FACx of ALS/MND donors could indicate senescence activation and cell cycle dysregulation in early stages of the disease.