Homocysteine metabolites inhibit autophagy, elevate amyloid beta, and induce neuropathy by impairing Phf8/H4K20me1-dependent epigenetic regulation of mTOR in cystathionine ß-synthase-deficient mice.

The loss of cystathionine ß-synthase (CBS), an important homocysteine (Hcy)-metabolizing enzyme or the loss of PHF8, an important histone demethylase participating in epigenetic regulation, causes severe intellectual disability in humans. Similar neuropathies were also observed in Cbs-/- and Phf8-/- mice. How CBS or PHF8 depletion can cause neuropathy was unknown. To answer this question, we examined a possible interaction between PHF8 and CBS using Cbs-/- mouse and neuroblastoma cell models. We quantified gene expression by RT-qPCR and western blotting, mTOR-bound H4K20me1 by chromatin immunoprecipitation (CHIP) assay, and amyloid ß (Aß) by confocal fluorescence microscopy using anti-Aß antibody. We found significantly reduced expression of Phf8, increased H4K20me1, increased mTOR expression and phosphorylation, and increased App, both on protein and mRNA levels in brains of Cbs-/- mice versus Cbs+/- sibling controls. Autophagy-related Becn1, Atg5, and Atg7 were downregulated while p62, Nfl, and Gfap were upregulated on protein and mRNA levels, suggesting reduced autophagy and increased neurodegeneration in Cbs-/- brains. In mouse neuroblastoma N2a or N2a-APPswe cells, treatments with Hcy-thiolactone, N-Hcy-protein or Hcy, or Cbs gene silencing by RNA interference significantly reduced Phf8 expression and increased total H4K20me1 as well as mTOR promoter-bound H4K20me1. This led to transcriptional mTOR upregulation, autophagy downregulation, and significantly increased APP and Aß levels. The Phf8 gene silencing increased Aß, but not APP, levels. Taken together, our findings identify Phf8 as a regulator of Aß synthesis and suggest that neuropathy of Cbs deficiency is mediated by Hcy metabolites, which transcriptionally dysregulate the Phf8 → H4K20me1 → mTOR → autophagy pathway thereby increasing Aß accumulation.

Sequence-non-specific effects generated by various types of RNA interference triggers.

RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition.

Homocysteine metabolites inhibit autophagy by upregulating miR-21-5p, miR-155-5p, miR-216-5p, and miR-320c-3p in human vascular endothelial cells.

Nutritional and genetic deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis, which is a major cause of cardiovascular disease (CVD). Impaired autophagy causes the accumulation of damaged proteins and organelles and is associated with CVD. Biochemically, HHcy is characterized by elevated levels of Hcy and its metabolites, Hcy-thiolactone and N-Hcy-protein. However, whether these metabolites can dysregulate mTOR signaling and autophagy in endothelial cells is not known. Here, we examined the influence of Hcy-thiolactone, N-Hcy-protein, and Hcy on autophagy human umbilical vein endothelial cells. We found that treatments with Hcy-thiolactone, N-Hcy-protein, or Hcy significantly downregulated beclin 1 (BECN1), autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 (LC3) mRNA and protein levels. We also found that these changes were mediated by upregulation by Hcy-thiolactone, N-Hcy-protein, and Hcy of autophagy-targeting microRNA (miR): miR-21, miR-155, miR-216, and miR-320c. The effects of these metabolites on levels of miR targeting autophagy as well as on the levels of BECN1, ATG5, ATG7, and LC3 mRNA and protein were abrogated by treatments with inhibitors of miR-21, miR-155, miR-216, and mir320c. Taken together, our findings show that Hcy metabolites can upregulate miR-21, miR-155, miR-216, and mir320c, which then downregulate autophagy in human endothelial cells, important for vascular homeostasis.

Diet-induced hyperhomocysteinemia causes sex-dependent deficiencies in offspring musculature and brain function.

Hyperhomocysteinemia (HHcy), characterized by elevated homocysteine (Hcy) levels, is a known risk factor for cardiovascular, renal, and neurological diseases, as well as pregnancy complications. Our study aimed to investigate whether HHcy induced by a high-methionine (high-Met) diet exacerbates cognitive and behavioral deficits in offspring and leads to other breeding problems. Dietary HHcy was induced four weeks before mating and continued throughout gestation and post-delivery. A battery of behavioral tests was conducted on offspring between postnatal days (PNDs) 5 and 30 to assess motor function/activity and cognition. The results were correlated with brain morphometric measurements and quantitative analysis of mammalian target of rapamycin (mTOR)/autophagy markers. The high-Met diet significantly increased parental and offspring urinary tHcy levels and influenced offspring behavior in a sex-dependent manner. Female offspring exhibited impaired cognition, potentially related to morphometric changes observed exclusively in HHcy females. Male HHcy pups demonstrated muscle weakness, evidenced by slower surface righting, reduced hind limb suspension (HLS) hanging time, weaker grip strength, and decreased activity in the beaker test. Western blot analyses indicated the downregulation of autophagy and the upregulation of mTOR activity in HHcy cortexes. HHcy also led to breeding impairments, including reduced breeding rate, in-utero fetal death, lower pups' body weight, and increased mortality, likely attributed to placental dysfunction associated with HHcy. In conclusion, a high-Met diet impairs memory and cognition in female juveniles and weakens muscle strength in male pups. These effects may stem from abnormal placental function affecting early neurogenesis, the dysregulation of autophagy-related pathways in the cortex, or epigenetic mechanisms of gene regulation triggered by HHcy during embryonic development.

Depletion of Paraoxonase 1 (Pon1) Dysregulates mTOR, Autophagy, and Accelerates Amyloid Beta Accumulation in Mice.

Paraoxonase 1 (PON1), a homocysteine (Hcy)-thiolactone detoxifying enzyme, has been associated with Alzheimer's disease (AD), suggesting that PON1 plays an important protective role in the brain. To study the involvement of PON1 in the development of AD and to elucidate the mechanism involved, we generated a new mouse model of AD, the Pon1-/-xFAD mouse, and examined how Pon1 depletion affects mTOR signaling, autophagy, and amyloid beta (Aß) accumulation. To elucidate the mechanism involved, we examined these processes in N2a-APPswe cells. We found that Pon1 depletion significantly downregulated Phf8 and upregulated H4K20me1; mTOR, phospho-mTOR, and App were upregulated while autophagy markers Bcln1, Atg5, and Atg7 were downregulated at the protein and mRNA levels in the brains of Pon1─/─5xFAD vs. Pon1+/+5xFAD mice. Pon1 depletion in N2a-APPswe cells by RNA interference led to downregulation of Phf8 and upregulation of mTOR due to increased H4K20me1-mTOR promoter binding. This led to autophagy downregulation and significantly increased APP and Aß levels. Phf8 depletion by RNA interference or treatments with Hcy-thiolactone or N-Hcy-protein metabolites similarly increased Aß levels in N2a-APPswe cells. Taken together, our findings define a neuroprotective mechanism by which Pon1 prevents Aß generation.

Deletion of the Homocysteine Thiolactone Detoxifying Enzyme Bleomycin Hydrolase, in Mice, Causes Memory and Neurological Deficits and Worsens Alzheimer's Disease-Related Behavioral and Biochemical Traits in the 5xFAD Model of Alzheimer's Disease.

BACKGROUND: Bleomycin hydrolase (BLMH), a homocysteine (Hcy)-thiolactone detoxifying enzyme, is attenuated in Alzheimer's disease (AD) brains. Blmh loss causes astrogliosis in mice while the loss of histone demethylase Phf8, which controls mTOR signaling, causes neuropathy in mice and humans. OBJECTIVE: To examine how Blmh gene deletion affects the Phf8/H4K20me1/mTOR/autophagy pathway, amyloid-ß (Aß) accumulation, and cognitive/neuromotor performance in mice. METHODS: We generated a new mouse model of AD, the Blmh-/-5xFAD mouse. Behavioral assessments were conducted by cognitive/neuromotor testing. Blmh and Phf8 genes were silenced in mouse neuroblastoma N2a-APPswe cells by RNA interference. mTOR- and autophagy-related proteins, and AßPP were quantified by western blotting and the corresponding mRNAs by RT-qPCR. Aß was quantified by western blotting (brains) and by confocal microscopy (cells). RESULTS: Behavioral testing showed cognitive/neuromotor deficits in Blmh-/- and Blmh-/-5xFAD mice. Phf8 was transcriptionally downregulated in Blmh-/- and Blmh-/-5xFAD brains. H4K20me1, mTOR, phospho-mTOR, and AßPP were upregulated while autophagy markers Becn1, Atg5, and Atg7 were downregulated in Blmh-/- and Blmh-/-5xFAD brains. Aß was elevated in Blmh-/-5xFAD brains. These biochemical changes were recapitulated in Blmh-silenced N2a-APPswe cells, which also showed increased H4K20me1-mTOR promoter binding and impaired autophagy flux (Lc3-I, Lc3-II, p62). Phf8-silencing or treatments with Hcy-thiolactone or N-Hcy-protein, metabolites elevated in Blmh-/- mice, induced biochemical changes in N2a-APPswe cells like those induced by the Blmh-silencing. However, Phf8-silencing elevated Aß without affecting AßPP. CONCLUSIONS: Our findings show that Blmh interacts with AßPP and the Phf8/H4K20me1/mTOR/autophagy pathway, and that disruption of those interactions causes Aß accumulation and cognitive/neuromotor deficits.

Neuroprotective Effects of Cranberry Juice Treatment in a Rat Model of Parkinson's Disease.

Rich in polyphenols, cranberry juice (CJ) with high antioxidant activity is believed to contribute to various health benefits. However, our knowledge of the neuroprotective potential of cranberries is limited. Previously, we have demonstrated that CJ treatment controls oxidative stress in several organs, with the most evident effect in the brain. In this study, we examined the capability of CJ for protection against Parkinson's disease (PD) in a rotenone (ROT) rat model. Wistar rats were administered with CJ in a dose of 500 mg/kg b.w./day (i.g.) and subcutaneously injected with ROT (1.3 mg/kg b.w./day). The experiment lasted 45 days, including 10 days pre-treatment with CJ and 35 days combined treatment with CJ and ROT. We quantified the expression of α-synuclein and apoptosis markers in the midbrain, performed microscopic examination, and assessed postural instability to evaluate the CJ neuroprotective effect. Our results indicate that the juice treatment provided neuroprotection, as evidenced by declined α-synuclein accumulation, Bax and cleaved/active caspase-9 expression, and normalized cytochrome c level that was accompanied by the enhancement of neuronal activity survival and improved postural instability. Importantly, we also found that long-term administration of CJ alone in a relatively high dose may exert a deleterious effect on cell survival in the midbrain.

Pomegranate Juice Ameliorates Dopamine Release and Behavioral Deficits in a Rat Model of Parkinson's Disease.

Pomegranate juice (PJ) is a rich source of ellagitannins (ETs), precursors of colonic metabolite urolithin A, which are believed to contribute to pomegranate's neuroprotective effect. While many experimental studies involving PJ's role in Alzheimer's disease and hypoxic-ischemic brain injury have been conducted, our knowledge of pomegranate's effects against Parkinson's disease (PD) is very limited. Previously, we have reported that PJ treatment improved postural stability, which correlated well with enhancement of neuronal survival, protection against oxidative damage, and α-synuclein aggregation. Since olfactory and motor deficits are typical symptoms of PD, in this study, we aimed to investigate the capability of PJ to protect against olfactory, motoric, and neurochemical alterations. To evaluate its efficiency, Wistar rats were given a combined treatment with ROT (1.3 mg/kg b.w./day, s.c.) and PJ (500 mg/kg/day, p.o.) for 35 days. After this, we assessed the olfactory discrimination index (DI) and vertical and horizontal activities as well as levels of dopamine and its main metabolite 3,4-Dihydroxyphenylacetic acid (DOPAC) in the dissected midbrain of animals. Our findings provide the first evidence that PJ treatment protects against ROT-induced DA depletion in the midbrain, which correlates well with improved olfactory function and vertical activity as well as with the presence of urolithin A in the brain.