Background: N6-methyladenosine (m⁶A) modification of RNA influences fundamental aspects of RNA metabolism and m⁶A dysregulation is implicated in various human diseases. In this study, we explored the potential role of RNA m⁶A modification in the pathogenesis of Alzheimer disease (AD).Methods: We investigated the m⁶A modification and the expression of m⁶A regulators in the brain tissues of AD patients and determined the impact and underlying mechanism of manipulated expression of m⁶A levels on AD-related deficits both in vitro and in vivo.Results: We found decreased neuronal m⁶A levels along with significantly reduced expression of m⁶A methyltransferase like 3 (METTL3) in AD brains. Interestingly, reduced neuronal m⁶A modification in the hippocampus caused by METTL3 knockdown led to significant memory deficits, accompanied by extensive synaptic loss and neuronal death along with multiple AD-related cellular alterations including oxidative stress and aberrant cell cycle events in vivo. Inhibition of oxidative stress or cell cycle alleviated shMettl3-induced apoptotic activation and neuronal damage in primary neurons. Restored m⁶A modification by inhibiting its demethylation in vitro rescued abnormal cell cycle events, neuronal deficits and death induced by METTL3 knockdown. Soluble Aβ oligomers caused reduced METTL3 expression and METTL3 knockdown exacerbated while METTL3 overexpression rescued Aβ-induced synaptic PSD95 loss in vitro. Importantly, METTL3 overexpression rescued Aβ-induced synaptic damage and cognitive impairment in vivo.Conclusions: Collectively, these data suggested that METTL3 reduction-mediated m⁶A dysregulation likely contributes to neurodegeneration in AD which may be a therapeutic target for AD.

In Alzheimer disease (AD), hyperphosphorylation of tau proteins results in microtubule destabilization and cytoskeletal abnormalities. Our prior ultra-morphometric studies documented a clear reduction in microtubules in pyramidal neurons in AD compared to controls, however, this reduction did not coincide with the presence of paired helical filaments. The latter suggests the presence of compensatory mechanism(s) that stabilize microtubule dynamics despite the loss of tau binding and stabilization. Microtubules are composed of tubulin dimers which are subject to posttranslational modifications that affect the stability and function of microtubules. In this study, we performed a detailed analysis on changes in the posttranslational modifications in tubulin in postmortem human brain tissues from AD patients and age-matched controls by immunoblot and immunocytochemistry. Consistent with our previous study, we found decreased levels of α-tubulin in AD brain. Levels of tubulin with various posttranslational modifications such as polyglutamylation, tyrosination, and detyrosination were also proportionally reduced in AD brain, but, interestingly, there was an increase in the proportion of the acetylated α-tubulin in the remaining α-tubulin. Tubulin distribution was changed from predominantly in the processes to be more accumulated in the cell body. The number of processes containing polyglutamylated tubulin was well preserved in AD neurons. While there was a cell autonomous detrimental effect of NFTs on tubulin, this is likely a gradual and slow process, and there was no selective loss of acetylated or polyglutamylated tubulin in NFT-bearing neurons. Overall, we suggest that the specific changes in tubulin modification in AD brain likely represent a compensatory response.

The human β-defensins (hBDs) are a highly conserved family of cationic antimicrobial and immunomodulatory peptides expressed primarily by epithelial cells in response to invasion by bacteria, fungi and some viruses. To date, the most studied members of this family of peptides are hBD-1, -2, and -3. Expression of hBD-1 and -2 has been demonstrated previously in cultured microglia and astrocytes of both mouse and human brain. Unlike inducible hBD-2 and -3, hBD-1 is constitutively expressed and is not generally upregulated by proinflammatory factors. In this study, we investigated whether hBDs, as active components of the innate immune response, are affected by pathological events in the Alzheimer’s disease (AD) brain. We assessed the expression of hBD-1, -2, and -3 in tissue obtained at autopsy from AD and age-matched control brains.

It is believed that mitochondrial fragmentation cause mitochondrial dysfunction and neuronal deficits in Alzheimer’s disease. We recently reported that constitutive knockout of the mitochondria fusion protein mitofusin2 (Mfn2) in the mouse brain causes mitochondrial fragmentation and neurodegeneration in the hippocampus and cortex. Here, we utilize an inducible mouse model to knock out Mfn2 (Mfn2 iKO) in adult mouse hippocampal and cortical neurons to avoid complications due to developmental changes. Electron microscopy shows the mitochondria become swollen with disorganized and degenerated cristae, accompanied by increased oxidative damage 8 weeks after induction, yet the neurons appear normal at the light level. At later timepoints, increased astrocyte and microglia activation appear and nuclei become shrunken and pyknotic. Apoptosis (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL) begins to occur at 9 weeks, and by 12 weeks, most hippocampal neurons are degenerated, confirmed by loss of NeuN. Prior to the loss of NeuN, aberrant cell-cycle events as marked by proliferating cell nuclear antigen (PCNA) and pHistone3 were evident in some Mfn2 iKO neurons but do not colocalize with TUNEL signals. Thus, this study demonstrated that Mfn2 ablation and mitochondrial fragmentation in adult neurons cause neurodegeneration through oxidative stress and neuroinflammation in vivo via both apoptosis and aberrant cell-cycle-event-dependent cell death pathways.

Background: Inflammation and oxidative stress are both involved in the pathogenesis of Alzheimer disease and have been shown to be reciprocally linked. One group of molecules that have been directly associated with inflammation and the production of free radicals are the prostaglandin 13,14-dihydro 15-keto PGF_2α and the isoprostane 8-iso-PGF_2α. Results: To further delineate the role of inflammatory and oxidative parameters in Alzheimer disease, in this study we evaluated the amount and localization of 13,14-dihydro 15-keto PGF_2α and 8-iso-PGF_2α in hippocampal post mortem tissue samples from age-matched Alzheimer disease and control patients. Our results demonstrate increased levels of 13,14-dihydro 15-keto PGF_2α and 8-iso-PGF_2α in the hippocampal pyramidal neurons of Alzheimer disease patients when compared to control patients. Conclusion: These data not only support the shared mechanistic involvement of free radical damage and inflammation in Alzheimer disease, but also indicate that multiple pathogenic "hits" are likely necessary for both the development and propagation of Alzheimer disease.

Background: Mutations in LRRK2 encoding leucine-rich repeat kinase 2 are thus far the most frequent genetic cause associated with autosomal dominant and idiopathic Parkinson's disease (PD). To examine whether LRRK2 is directly associated with neuropathology of PD and other related disorders, we analyzed LRRK2 in brains of patients affected by PD and dementia with Lewy bodies (DLB) using highly specific antibodies to LRRK2. Results: We demonstrated that anti-LRRK2 antibodies strongly labelled brainstem and cortical Lewy bodies, the pathological hallmarks of PD and DLB, respectively. In addition, anti-LRRK2 also labelled brain vasculature, axons, and neuronal cell bodies. Interestingly, the immunocytochemical profile of LRRK2 varied with different antibodies depending upon specific antigenic sites along the LRRK2 protein. All anti-LRRK2 antibodies tested that were raised against various regions of LRRK2, were found to be immunoreactive to recombinant LRRK2 on Western blots. However, only the antibodies raised against the N-terminal and C-terminal regions of LRRK2, but not the regions containing folded protein domains, were positive in immunolabeling of Lewy bodies, suggesting a differential exposure of specific antigenic sites of LRRK2 on tissue sections. Conclusion: We conclude that LRRK2 is a component of Lewy bodies in both PD and DLB, and therefore plays an important role in the Lewy body formation and disease pathogenesis. Information on the cellular localization of LRRK2 under normal and pathological conditions will deepen our understanding of its functions and molecular pathways relevant to the progression of PD and related disorders.