Maria Manczak

Protective effects of reduced dynamin-related protein 1 against amyloid beta-induced mitochondrial dysfunction and synaptic damage in Alzheimer's disease

The purpose of our study was to understand the protective effects of reduced expression of dynamin-related protein (Drp1) against amyloid beta (Aβ) induced mitochondrial and synaptic toxicities in Alzheimers disease (AD) progression and pathogenesis. Our recent molecular and biochemical studies revealed that impaired mitochondrial dynamics-increased mitochondrial fragmentation and decreased fusion-in neurons from autopsy brains of AD patients and from transgenic AD mice and neurons expressing Aβ, suggesting that Aβ causes mitochondrial fragmentation in AD. Further, our recent co-immunoprecipitation and immunostaining analysis revealed that the mitochondrial fission protein Drp1 interacted with Aβ, and this interaction increased as AD progressed. Based on these findings, we hypothesize that a partial deficiency of Drp1 inhibits Drp1-Aβ interactions and protects Aβ-induced mitochondrial and synaptic toxicities, and maintains mitochondrial dynamics and neuronal function in AD neurons. We crossed Drp1+/- mice with APP transgenic mice (Tg2576 line) and created double mutant (APPXDrp1+/-) mice. Using real-time RT-PCR and immunoblotting analyses, we measured mRNA expressions and protein levels of genes related to the mitochondrial dynamics, mitochondrial biogenesis and synapses from 6-month-old Drp1+/-, APP, APPXDrp1+/- and wild-type (WT) mice. Using biochemical methods, we also studied mitochondrial function and measured soluble Aβ in brain tissues from all lines of mice in our study. Decreased mRNA expressions and protein levels of Drp1 and Fis1 (fission) and CypD (matrix) genes, and increased levels of Mfn1, Mfn2 and Opa1 (fusion), Nrf1, Nrf2, PGC1α, TFAM (biogenesis) and synaptophysin, PSD95, synapsin 1, synaptobrevin 1, neurogranin, GAP43 and synaptopodin (synaptic) were found in 6-month-old APPXDrp1+/- mice relative to APP mice. Mitochondrial functional assays revealed that mitochondrial dysfunction is reduced in APPXDrp1+/- mice relative to APP mice, suggesting that reduced Drp1enhances mitochondrial function in AD neurons. Sandwich ELISA assay revealed that soluble Aβ levels were significantly reduced in APPXDrp1+/- mice relative to APP mice, indicating that reduced Drp1 decreases soluble Aβ production in AD progression. These findings suggest that a partial reduction of Drp1 reduces Aβ production, reduces mitochondrial dysfunction, and maintains mitochondrial dynamics, enhances mitochondrial biogenesis and synaptic activity in APP mice. These findings may have implications for the development of Drp1 based therapeutics for AD patients.

Reduced dynamin-related protein 1 protects against phosphorylated Tau-induced mitochondrial dysfunction and synaptic damage in Alzheimer’s disease

The purpose of our study was to understand the protective effects of a partial reduction of dynamin-related protein 1 (Drp1) in Alzheimer’s disease (AD) progression and pathogenesis. Increasing evidence suggests that phosphorylated Tau and mitochondrial abnormalities are involved in the loss of synapses, defective axonal transport and cognitive decline, in patients with AD. In the current study, we investigated whether a partial reduction of Drp1 protect neurons from phosphorylated Tau-induced mitochondrial and synaptic toxicities in AD progression. We crossed Drp1+/− mice with Tau transgenic mice (P301L line) and created double mutant (TauXDrp1+/−) mice. Using real-time RT-PCR, immunoblotting and immunostaining analyses, we measured mRNA expressions and protein levels of genes related to the mitochondrial dynamics—Drp1 and Fis1 (fission), Mfn1, Mfn2 and Opa1 (fusion), CypD (matrix), mitochondrial biogenesis—Nrf1, Nrf2, PGC1α and TFAM and synaptic—synaptophysin, PSD95, synapsin 1, synaptobrevin 1, neurogranin, GAP43 and synaptopodin in brain tissues from 6-month-old Drp1+/−, Tau, TauXDrp1+/− and wild-type mice. Using biochemical and immunoblotting methods, mitochondrial function and phosphorylated Tau were measured. Decreased mRNA and protein levels of fission and matrix and increased levels of fusion, mitochondrial biogenesis, and synaptic genes were found in 6-month-old TauXDrp1+/− mice relative to Tau mice. Mitochondrial dysfunction was reduced in TauXDrp1+/− mice relative to Tau mice. Phosphorylated Tau found to be reduced in TauXDrp1+/− mice relative to Tau mice. These findings suggest that a partial reduction of Drp1 decreases the production of phosphorylated Tau, reduces mitochondrial dysfunction, and maintains mitochondrial dynamics, enhances mitochondrial biogenesis and synaptic activity in Tau mice. Findings of this study may have implications for the development of Drp1 based therapeutics for patients with AD and other tauopathies.

Hippocampal phosphorylated tau induced cognitive decline, dendritic spine loss and mitochondrial abnormalities in a mouse model of Alzheimer's disease

The purpose of our study was to understand the toxic effects of hippocampal phosphorylated tau in tau mice. Using rotarod and Morris water maze (MWM) tests, immunoblotting and immunofluorescence, Golgi-Cox staining and transmission electron microscopy, we assessed cognitive behavior, measured protein levels of mitochondrial dynamics, MAP2, total and phosphorylated tau, and quantified dendritic spines and mitochondrial number and length in 12-month-old tau mice with P301L mutation. Mitochondrial function was assessed by measuring the levels of H2O2, lipid peroxidation, cytochrome oxidase activity and mitochondrial ATP. MWM and rotarod tests revealed that hippocampal learning and memory and motor learning and coordination were impaired in tau mice relative to wild-type (WT) mice. Increased levels of mitochondrial fission proteins, Drp1 and Fis1 and decreased levels of mitochondrial fusion proteins, Mfn1, Mfn2 and Opa1 were found in 12-month-old tau mice relative to age-matched WT mice, indicating that the presence of abnormal mitochondrial dynamics in tau mice. Decreased levels of dendritic protein, MAP2 and increased levels of total and phosphorylated tau proteins were found in tau mice relative to WT mice. Mitochondrial function was defective. Golgi-Cox staining analysis revealed that dendritic spines are significantly reduced. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in tau mice. These findings suggest that hippocampal accumulation of phosphorylated tau is responsible for abnormal mitochondrial dynamics and reducing dendritic protein MAP2 and dendritic spines and hippocampal based learning and memory impairments, and mitochondrial structural and functional changes in tau mice. Based on these observations, we propose that reduced hippocampal phosphorylated tau is an important therapeutic strategy for AD and other tauopathies.

Protective effects of a natural product, curcumin, against amyloid β induced mitochondrial and synaptic toxicities in Alzheimer's disease

The purpose of our study was to investigate the protective effects of a natural product—‘curcumin’— in Alzheimers disease (AD)-like neurons. Although much research has been done in AD, very little has been reported on the effects of curcumin on mitochondrial biogenesis, dynamics, function and synaptic activities. Therefore, the present study investigated the protective effects against amyloid β (Aβ) induced mitochondrial and synaptic toxicities. Using human neuroblastoma (SHSY5Y) cells, curcumin and Aβ, we studied the protective effects of curcumin against Aβ. Further, we also studied preventive (curcumin+Aβ) and intervention (Aβ+curcumin) effects of curcumin against Aβ in SHSY5Y cells. Using real time RT-PCR, immunoblotting and immunofluorescence analysis, we measured mRNA and protein levels of mitochondrial dynamics, mitochondrial biogenesis and synaptic genes. We also assessed mitochondrial function by measuring hydrogen peroxide, lipid peroxidation, cytochrome oxidase activity and mitochondrial ATP. Cell viability was studied using the MTT assay. Aβ was found to impair mitochondrial dynamics, reduce mitochondrial biogenesis and decrease synaptic activity and mitochondrial function. In contrast, curcumin enhanced mitochondrial fusion activity and reduced fission machinery, and increased biogenesis and synaptic proteins. Mitochondrial function and cell viability were elevated in curcumin treated cells. Interestingly, curcumin pre- and post-treated cells incubated with Aβ showed reduced mitochondrial dysfunction, and maintained cell viability and mitochondrial dynamics, mitochondrial biogenesis and synaptic activity. Further, the protective effects of curcumin were stronger in pretreated SHSY5Y cells than in post-treated cells, indicating that curcumin works better in prevention than treatment in AD-like neurons. Our findings suggest that curcumin is a promising drug molecule to treat AD patients.

Mitochondria-targeted small molecule SS31: a potential candidate for the treatment of Alzheimer’s disease

The objective of our study was to better understand the protective effects of the mitochondria-targeted tetra-peptide SS31 against amyloid beta (Aβ)-induced mitochondrial and synaptic toxicities in Alzheimers disease (AD) progression. Using intraperitoneal injections, we administered SS31 to an AD mouse model (APP) over a period of 6 weeks, beginning when the APP mice were 12 months of age. We studied their cortical tissues after SS31 treatment and determined that SS31 crosses the blood brain barrier and reaches mitochondrial sites of free radical production. We also determined: (1) plasma and brain levels of SS31, (2) mRNA levels and levels of mitochondrial dynamics, biogenesis proteins and synaptic proteins, (3) soluble Aβ levels and immunoreactivity of mutant APP and Aβ levels and (4) mitochondrial function by measuring H2O2, lipid peroxidation, cytochrome c oxidase activity and mitochondrial ATP. We found reduced mRNA expression and reduced protein levels of fission genes, and increased levels of mitochondrial fusion, biogenesis and synaptic genes in SS31-treated APP mice relative to SS31-untreated APP mice. Immunofluorescence analysis revealed reduced full-length mutant APP and soluble/insoluble Aβ levels in the SS31-treated APP mice. Sandwich ELISA assays revealed significantly reduced soluble Aβ levels in the SS31-treated APP mice relative to the untreated APP mice. Mitochondrial function was maintained in the SS31-treated APP mice over the 6 weeks of SS31 treatment compared with mitochondrial function in the untreated APP mice. Our findings indicate that SS31 treatment reduces Aβ production, reduces mitochondrial dysfunction, maintains mitochondrial dynamics and enhances mitochondrial biogenesis and synaptic activity in APP mice; and that SS31 may confer protective effects against mitochondrial and synaptic toxicities in APP transgenic mice.

Hippocampal mutant APP and amyloid beta-induced cognitive decline, dendritic spine loss, defective autophagy, mitophagy and mitochondrial abnormalities in a mouse model of Alzheimer’s disease

The purpose of our study was to determine the toxic effects of hippocampal mutant APP and amyloid beta (Aβ) in 12-month-old APP transgenic mice. Using rotarod and Morris water maze tests, immunoblotting and immunofluorescence, Golgi-cox staining and transmission electron microscopy, we assessed cognitive behavior, protein levels of synaptic, autophagy, mitophagy, mitochondrial dynamics, biogenesis, dendritic protein MAP2 and quantified dendritic spines and mitochondrial number and length in 12-month-old APP mice that express Swedish mutation. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity and mitochondrial ATP. Morris water maze and rotarod tests revealed that hippocampal learning and memory and motor learning and coordination were impaired in APP mice relative to wild-type (WT) mice. Increased levels of mitochondrial fission proteins, Drp1 and Fis1 and decreased levels of fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1α, NRF1, NRF2 and TFAM), autophagy (ATG5 and LC3BI, LC3BII), mitophagy (PINK1 and TERT), synaptic (synaptophysin and PSD95) and dendritic (MAP2) proteins were found in 12-month-old APP mice relative to age-matched non-transgenic WT mice. Golgi-cox staining analysis revealed that dendritic spines are significantly reduced. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in APP mice. These findings suggest that hippocampal accumulation of mutant APP and Aβ is responsible for abnormal mitochondrial dynamics and defective biogenesis, reduced MAP2, autophagy, mitophagy and synaptic proteins and reduced dendritic spines and hippocampal-based learning and memory impairments, and mitochondrial structural and functional changes in 12-month-old APP mice.

Protective Effects of Indian Spice Curcumin Against Amyloid-β in Alzheimer’s Disease

The purpose of our article is to assess the current understanding of Indian spice, curcumin, against amyloid-β (Aβ)-induced toxicity in Alzheimers disease (AD) pathogenesis. Natural products, such as ginger, curcumin, and gingko biloba have been used as diets and dietary supplements to treat human diseases, including cancer, cardiovascular, respiratory, infectious, diabetes, obesity, metabolic syndromes, and neurological disorders. Products derived from plants are known to have protective effects, including anti-inflammatory, antioxidant, anti-arthritis, pro-healing, and boosting memory cognitive functions. In the last decade, several groups have designed and synthesized curcumin and its derivatives and extensively tested using cell and mouse models of AD. Recent research on Aβ and curcumin has revealed that curcumin prevents Aβ aggregation and crosses the blood-brain barrier, reach brain cells, and protect neurons from various toxic insults of aging and Aβ in humans. Recent research has also reported that curcumin ameliorates cognitive decline and improves synaptic functions in mouse models of AD. Further, recent groups have initiated studies on elderly individuals and patients with AD and the outcome of these studies is currently being assessed. This article highlights the beneficial effects of curcumin on AD. This article also critically assesses the current limitations of curcumins bioavailability and urgent need for new formulations to increase its brain levels to treat patients with AD.

MicroRNAs, Aging, Cellular Senescence, and Alzheimer's Disease

Aging is a normal process of living being. It has been reported that multiple cellular changes, including oxidative damage/mitochondrial dysfunction, telomere shortening, inflammation, may accelerate the aging process, leading to cellular senescence. These cellular changes induce age-related human diseases, including Alzheimers, Parkinsons, multiple sclerosis, amyotrophic lateral sclerosis, cardiovascular, cancer, and skin diseases. Changes in somatic and germ-line DNA and epigenetics are reported to play large roles in accelerating the onset of human diseases. Cellular mechanisms of aging and age-related diseases are not completely understood. However, recent discoveries in molecular biology have revealed that microRNAs (miRNAs) are potential indicators of aging, cellular senescence, and Alzheimers disease (AD). The purpose of our chapter is to highlight recent advancements in miRNAs and their involvement in cellular changes in aging, cellular senescence, and AD. This chapter also critically evaluates miRNA-based therapeutic drug targets for aging and age-related diseases, particularly Alzheimers.

Mutant APP and amyloid beta-induced defective autophagy, mitophagy, mitochondrial structural and functional changes and synaptic damage in hippocampal neurons from Alzheimer’s disease

The purpose of our study was to determine the toxic effects of hippocampal mutant APP (mAPP) and amyloid beta (Aβ) in human mAPP complementary DNA (cDNA) transfected with primary mouse hippocampal neurons (HT22). Hippocampal tissues are the best source of studying learning and memory functions in patients with Alzheimers disease (AD) and healthy controls. However, investigating immortalized hippocampal neurons that express AD proteins provide an excellent opportunity for drug testing. Using quantitative reverse transcriptase-polymerase chain reaction, immunoblotting & immunofluorescence and transmission electron microscopy, we assessed messenger RNA (mRNA) and protein levels of synaptic, autophagy, mitophagy, mitochondrial dynamics, biogenesis, dendritic protein MAP2 and assessed mitochondrial number and length in mAPP-HT22 cells that express Swedish/Indiana mutations. Mitochondrial function was assessed by measuring the levels of hydrogen peroxide, lipid peroxidation, cytochrome c oxidase activity and mitochondrial adenosine triphosphate. Increased levels of mRNA and protein levels of mitochondrial fission genes, Drp1 and Fis1 and decreased levels fusion (Mfn1, Mfn2 and Opa1) biogenesis (PGC1α, NRF1, NRF2 & TFAM), autophagy (ATG5 & LC3BI, LC3BII), mitophagy (PINK1 & TERT, BCL2 & BNIPBL), synaptic (synaptophysin & PSD95) and dendritic (MAP2) genes were found in mAPP-HT22 cells relative to WT-HT22 cells. Cell survival was significantly reduced mAPP-HT22 cells. GTPase-Drp1 enzymatic activity was increased in mAPP-HT22 cells. Transmission electron microscopy revealed significantly increased mitochondrial numbers and reduced mitochondrial length in mAPP-HT22 cells. These findings suggest that hippocampal accumulation of mAPP and Aβ is responsible for abnormal mitochondrial dynamics and defective biogenesis, reduced MAP2, autophagy, mitophagy and synaptic proteins & reduced dendritic spines and mitochondrial structural and functional changes in mAPP hippocampal cells. These observations strongly suggest that accumulation of mAPP and Aβ causes mitochondrial, synaptic and autophagy/mitophagy abnormalities in hippocampal neurons, leading to neuronal dysfunction.

Maternal exercise upregulates mitochondrial gene expression and increases enzyme activity of fetal mouse hearts

Maternal exercise during pregnancy has been shown to improve the long‐term health of offspring in later life. Mitochondria are important organelles for maintaining adequate heart function, and mitochondrial dysfunction is linked to cardiovascular disease. However, the effects of maternal exercise during pregnancy on mitochondrial biogenesis in hearts are not well understood. Thus, the purpose of this study was to test the hypothesis that mitochondrial gene expression in fetal myocardium would be upregulated by maternal exercise. Twelve‐week‐old female C57BL/6 mice were divided into sedentary and exercise groups. Mice in the exercise group were exposed to a voluntary cage‐wheel from gestational day 1 through 17. Litter size and individual fetal weights were taken when pregnant dams were sacrificed at 17 days of gestation. Three to four hearts from the same group were pooled to study gene expression, protein expression, and enzyme activity. There were no significant differences in litter size, sex distribution, and average fetal body weight per litter between sedentary and exercised dams. Genes encoding mitochondrial biogenesis and dynamics, including nuclear respiratory factor‐1 (Nrf1), Nrf2, and dynamin‐related GTPase termed mitofusin‐2 (Mfn2) were significantly upregulated in the fetal hearts from exercised dams. Cytochrome c oxidase activity and ATP production were significantly increased, while the hydrogen peroxide level was significantly decreased in the fetal hearts by maternal exercise. Our results demonstrate that maternal exercise initiated at day 1 of gestation could transfer the positive mitochondrial phenotype to fetal hearts.