Dorsal Root Ganglia Mitochondrial Biochemical Changes in Non-diabetic and Streptozotocin-Induced Diabetic Mice Fed with a Standard or High-Fat Diet.

BACKGROUND: Mitochondrial dysfunction is purported as a contributory mechanism underlying diabetic neuropathy, but a defined role for damaged mitochondria in diabetic nerves remains unclear, particularly in standard diabetes models. Experiments here used a high-fat diet in attempt to exacerbate the severity of diabetes and expedite the time-course in which mitochondrial dysfunction may occur. We hypothesized a high-fat diet in addition to diabetes would increase stress on sensory neurons and worsen mitochondrial dysfunction. METHODS: Oxidative phosphorylation proteins and proteins associated with mitochondrial function were quantified in lumbar dorsal root ganglia. Comparisons were made between non-diabetic and streptozotocin-induced (STZ) C57Bl/6 mice fed a standard or high-fat diet for 8 weeks. RESULTS: Complex III subunit Core-2 and voltage dependent anion channel were increased (by 36% and 28% respectively, p<0.05) in diabetic mice compared to nondiabetic mice fed the standard diet. There were no differences among groups in UCP2, PGC-1α, PGC-1ß levels or Akt, mTor, or AMPK activation. These data suggest compensatory mitochondrial biogenesis occurs to offset potential mitochondrial dysfunction after 8 weeks of STZ-induced diabetes, but a high-fat diet does not alter these parameters. CONCLUSION: Our results indicate mitochondrial protein changes early in STZ-induced diabetes. Interestingly, a high-fat diet does not appear to affect mitochondrial proteins in either nondiabetic or STZ- diabetic mice.

Mitochondria, Cybrids, Aging, and Alzheimer's Disease.

Mitochondrial and bioenergetic function change with advancing age and may drive aging phenotypes. Mitochondrial and bioenergetic changes are also documented in various age-related neurodegenerative diseases, including Alzheimer's disease (AD). In some instances AD mitochondrial and bioenergetic changes are reminiscent of those observed with advancing age but are greater in magnitude. Mitochondrial and bioenergetic dysfunction could, therefore, link neurodegeneration to brain aging. Interestingly, mitochondrial defects in AD patients are not brain-limited, and mitochondrial function can be linked to classic AD histologic changes including amyloid precursor protein processing to beta amyloid. Also, transferring mitochondria from AD subjects to cell lines depleted of endogenous mitochondrial DNA (mtDNA) creates cytoplasmic hybrid (cybrid) cell lines that recapitulate specific biochemical, molecular, and histologic AD features. Such findings have led to the formulation of a "mitochondrial cascade hypothesis" that places mitochondrial dysfunction at the apex of the AD pathology pyramid. Data pertinent to this premise are reviewed.

Elevated reactive oxygen species and antioxidant enzyme activities in animal and cellular models of Parkinson's disease.

The dopaminergic neurotoxin N-methyl,4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) causes a syndrome in primates and humans which mimics Parkinson's disease (PD) in clinical, pathological, and biochemical findings, including diminished activity of complex I in the mitochondrial electron transport chain. Reduced complex I activity is found in sporadic PD and can be transferred through mitochondrial DNA, suggesting a mitochondrial genetic etiology. We now show that MPTP treatment of mice and N-methylpyridinium (MPP+) exposure of human SH-SY5Y neuroblastoma cells increases oxygen free radical production and antioxidant enzyme activities. Cybrid cells created by transfer of PD mitochondria exhibit similar characteristics; however, PD cybrids' antioxidant enzyme activities are not further increased by MPP+ exposure, as are the activities in control cybrids. PD mitochondrial cybrids are subject to metabolic and oxidative stresses similar to MPTP parkinsonism and provide a model to determine mechanisms of oxidative damage and cell death in PD.

Cybrids in Alzheimer's disease: a cellular model of the disease?

The mitochondrial electron transport chain enzyme cytochrome c oxidase (COX) is defective in patients with sporadic Alzheimer's disease (AD). This defect arises from the mutation of mitochondrial DNA (mtDNA). To develop a tissue culture system that would express this genetically derived bioenergetic lesion and permit characterization of its functional consequences, we depleted Ntera2/D1 (NT2) teratocarcinoma cells of endogenous mtDNA and repopulated them with platelet mtDNA from AD patients. Cytochrome c oxidase activity was depressed in the resulting AD cytoplasmic hybrids (cybrids) compared with cybrids prepared with mtDNA from non-AD controls. Reactive oxygen species (ROS) production and free radical scavenging enzyme activities were significantly elevated in AD cybrids. A COX defect in NT2 AD cybrid lines indicates that AD patients possess mtDNA COX gene mutations that are sufficient for determining this biochemical lesion. Expression of unique functional characteristics (increased ROS production and free radical scavenging enzyme activities) relevant to neurodegeneration demonstrates the utility of these cells in defining AD pathophysiology at a cellular level. This in vitro tissue culture model of AD may prove useful in drug screening.

Use of cytoplasmic hybrid cell lines for elucidating the role of mitochondrial dysfunction in Alzheimer's disease and Parkinson's disease.

There is substantial evidence of mitochondrial defects in neurodegenerative disorders such as Alzheimer's and Parkinson's diseases (AD and PD). We have probed the molecular implications of mitochondrial dysfunction in these diseases by transferring mitochondria from platelets obtained from disease and control donors into mitochondrial DNA-depleted recipient neuron-based cells (rho 0 cells). This process creates cytoplasmic hybrid (cybrid) cells where the mitochondrial DNA (mtDNA) from the donor is expressed in the nuclear and cellular background of the host rho 0 cell. Differences in phenotype between disease and control groups can thus be attributed to the exogenous mitochondria and mtDNA. Key methodological issues relating to this approach were addressed by demonstrating that recipient rho 0 cells have < 1 mtDNA copy/cell, and that exclusive repopulation with donor mtDNA occurs in cybrid cells. Further, we describe that sampling of heterogeneous cell populations is a valid approach for cybrid analysis. Our studies show that the focal respiratory chain defects reported in platelets of AD and PD cybrids can be recapitulated in AD and PD cybrids. In addition, both AD and PD cybrids display increased oxidative stress and perturbations in calcium homeostasis. These data suggest that the transfer of a mtDNA defect from disease donor platelets is the likely cause of the cybrid biochemical phenotype, and highlight the potential value of these cell lines as cellular disease models.

Reduction of MPP(+)-induced hydroxyl radical formation and nigrostriatal MPTP toxicity by inhibiting nitric oxide synthase.

N-Methyl, 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces experimental parkinsonism after oxidation to N-methylpyridinium ion (MPP+), accumulation in dopamine neurons and concentration in mitochondria. Inhibition by MPP+ of mitochondrial electron transport impairs respiratory function, but the molecular mechanisms of cell death are not clear. We tested the hypothesis that locally produced nitric oxide is a key component in MPTP toxicity by providing a necessary intermediate in the production of hydroxyl free radicals. Inhibition of nitric oxide synthase reduced MPP(+)-induced hydroxyl radical formation in striatum and MPTP toxicity to nigrostriatal dopamine terminals, but did not interfere with inhibition of complex-I activity. Nitric oxide appears to be necessary for hydroxyl free radical generation in MPP+ toxicity and may play a role in neuronal degeneration in Parkinson's disease.

Is NADH effective in the treatment of Parkinson's disease?

Most Parkinson's disease (PD) treatments palliate symptoms by increasing nigrostriatal dopaminergic tone. A unique strategy for accomplishing this pharmacological end-point proposes using reduced nicotinamide adenine dinucleotide (NADH) to boost endogenous dopamine production, since NADH indirectly supplies reducing equivalents to the rate-limiting, tyrosine hydroxylase-catalysed step of dopamine synthesis. Support for using NADH in PD treatment includes claims that NADH stimulates tyrosine hydroxylase and dopamine biosynthesis in tissue culture and humans, as well as case series associating intravenous and oral NADH administration with PD rating scale improvements. Theoretical and practical arguments against NADH include underlying NADH disposal impairment in PD and failure of a placebo-controlled trial to show any clear benefit. While NADH may yet prove to ameliorate parkinsonism, recommendations for its use in PD are premature.

Reduced gray matter volume in normal adults with a maternal family history of Alzheimer disease.

OBJECTIVE: A consistently identified risk factor for Alzheimer disease (AD) is family history of dementia, with maternal transmission significantly more frequent than paternal transmission. A history of maternal AD may be related to AD-like glucose consumption in cognitively healthy subjects. In this cross-sectional study, we tested whether cognitively healthy people with a family history of AD have less gray matter volume (GMV), an endophenotype for late-onset AD, than individuals with no family history, and whether decreases in GMV are different in subjects with a maternal family history. METHODS: As part of the Kansas University Brain Aging Project, 67 cognitively intact individuals with a maternal history of late-onset AD (FHm, n = 16), a paternal history of AD (FHp, n = 8), or no parental history of AD (FH-, n = 43), similar in age, gender, education, and Mini-Mental State Examination score, were scanned at 3 T. We used voxel-based morphometry to examine GMV differences between groups, controlling for age, gender, and apoE4. RESULTS: Cognitively healthy individuals with a family history of late-onset AD had significantly decreased GMV in the precuneus, middle frontal, inferior frontal, and superior frontal gyri compared with FH- individuals. FHm subjects had significantly smaller inferior frontal, middle frontal, precuneus, and lingual gyri compared with FH- and FHp subjects. CONCLUSIONS: Overall, maternal family history of Alzheimer disease (AD) in cognitively normal individuals is associated with lower gray matter volume in AD-vulnerable brain regions. These data complement and extend reports of cerebral metabolic differences in subjects with a maternal family history.

Autosomal dominant subcortical gliosis presenting as frontotemporal dementia.

OBJECTIVE: To describe a multigenerational kindred with a frontotemporal dementia clinical syndrome (FTDS), extensive subcortical gliosis pathology, and autosomal dominant genetics. METHODS: Clinical, imaging, and pathologic evaluations of multiple family members. RESULTS: Symptom onset commonly occurred in the fifth or sixth decade, although some kindred members did not develop obvious symptoms until their eighth decade. White matter changes were prominent on both MRI and CT imaging. Results from six brain autopsy evaluations showed consistent but varying degrees of pathology that, while unique, share some histologic similarities with leukodystrophies. These brains were notably devoid of both tau- and ubiquitin-containing inclusions. CONCLUSIONS: Subcortical gliosis in this kindred arises from mutation of a novel gene or else represents a unique frontotemporal dementia clinical syndrome variant caused by mutation of an already known gene. Clinical relevance and research implications are discussed.

Cardiorespiratory fitness and brain atrophy in early Alzheimer disease.

OBJECTIVE: To examine the correlation of cardiorespiratory fitness with brain atrophy and cognition in early-stage Alzheimer disease (AD). BACKGROUND: In normal aging physical fitness appears to mitigate functional and structural age-related brain changes. Whether this is observed in AD is not known. METHODS: Subjects without dementia (n = 64) and subjects with early-stage AD (n = 57) had MRI and standard clinical and psychometric evaluations. Peak oxygen consumption (VO(2)(peak)), the standard measure of cardiorespiratory fitness, was assessed during a graded treadmill test. Normalized whole brain volume, a brain atrophy estimate, was determined by MRI. Pearson correlation and linear regression were used to assess fitness in relation to brain volume and cognitive performance. RESULTS: Cardiorespiratory fitness (VO(2)(peak)) was modestly reduced in subjects with AD (34.7 [5.0] mL/kg/min) vs subjects without dementia (38.1 [6.3] mL/kg/min, p = 0.002). In early AD, VO(2)(peak) was associated with whole brain volume (beta = 0.35, p = 0.02) and white matter volume (beta = 0.35, p = 0.04) after controlling for age. Controlling for additional covariates of sex, dementia severity, physical activity, and physical frailty did not attenuate the relationships. VO(2)(peak) was associated with performance on delayed memory and digit symbol in early AD but not after controlling for age. In participants with no dementia, there was no relationship between fitness and brain atrophy. Fitness in participants with no dementia was associated with better global cognitive performance (r = 0.30, p = 0.02) and performance on Trailmaking A and B, Stroop, and delayed logical memory but not after controlling for age. CONCLUSIONS: Increased cardiorespiratory fitness is associated with reduced brain atrophy in Alzheimer disease (AD). Cardiorespiratory fitness may moderate AD-related brain atrophy or a common underlying AD-related process may impact both brain atrophy and cardiorespiratory fitness.

A novel deafness/dystonia peptide gene mutation that causes dystonia in female carriers of Mohr-Tranebjaerg syndrome.

Sex-linked male deafness and dystonia (Mohr-Tranebjaerg syndrome) arises from mutation of the deafness/dystonia peptide (DDP) gene. We describe a novel guanine deletion at nucleotide 108 of the DDP gene in a family with Mohr-Tranebjaerg syndrome, which terminates this 97-amino acid protein at codon 25. Unlike previously reported kindreds, carrier females in this family also manifest dystonias, including torticollis and writer's cramp. A family history of male deafness should alert clinicians to the possibility of DDP mutation in women with focal dystonias.

Mitochondrial dysfunction in idiopathic Parkinson disease.

Disordered mitochondrial metabolism may play an important role in a number of idiopathic neurodegenerative disorders. The question of mitochondrial dysfunction is particularly attractive in the case of idiopathic Parkinson disease (PD), since Vyas et al. recognized in the 1980s that the parkinsonism-inducing compound N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is a mitochondrial toxin. The unique genetic properties of mitochondria also make them worthy of consideration for a pathogenic role in PD, as well as in other late-onset, sporadic neurodegenerative disorders. Although affected persons occasionally do provide family histories that suggest Mendelian inheritance, the vast majority of the time these diseases appear sporadically. Because of unique features such as heteroplasmy, replicative segregation, and threshold effects, mitochondrial inheritance can allow for the apparent sporadic nature of these diseases.

Behcet's disease: presentation with sagittal sinus thrombosis diagnosed noninvasively.

The standard evaluation of patients with intracranial hypertension frequently does not reveal a discrete pathophysiologic process, leading in these cases to classification of the syndrome as "benign." We present a 35-year-old woman with a recent diagnosis of pseudotumor cerebri who presented with headache, emesis, and blurring of vision. Her symptoms were progressive despite two lumbar punctures that revealed normal cerebrospinal fluid under high pressure. Contrast and noncontrast CT scans were normal; both the cerebrospinal fluid and CT neuroimaging were thus consistent with benign intracranial hypertension. An MRI, however, supported the presence of sagittal sinus thrombosis, a finding which was confirmed by MR venography. Further workup for an underlying cause of sinus thrombosis disclosed symptoms and signs fulfilling the diagnostic criteria for Behcet's disease. Cerebral venous (or sinus) thrombosis should be considered in the differential diagnosis of intracranial hypertension. Behcet's disease, while extremely rare, should be considered as a potential cause of cerebral venous thrombosis. Magnetic resonance venography can serve as a useful diagnostic study in situations where confirmation or exclusion of sinus thrombosis is required.

Role of mitochondria in amyotrophic lateral sclerosis.

Neurodegeneration in amyotrophic lateral sclerosis (ALS) is characterized by the specific loss of central and peripheral motor neurons. While this pattern of neuronal demise gives rise to a distinct clinical syndrome, at the cellular and molecular level ALS pathology is similar to that seen in other neurodegenerative diseases. In particular, mitochondrial dysfunction in ALS is reminiscent of that observed in Alzheimer's and Parkinson's diseases. Mitochondria in persons with ALS demonstrate impaired electron transport, increased free radical generation, and an inability to adequately buffer cytosolic calcium shifts. These abnormalities are probably systemic and potentially due to mutation of mitochondrial DNA.

Altered calcium homeostasis in cells transformed by mitochondria from individuals with Parkinson's disease.

Parkinson's disease may be linked to defects in mitochondrial function. Mitochondrially transformed cells (cybrids) were created from Parkinson's disease patients or disease-free controls. Parkinson's disease cybrids had 26% less complex I activity, but maintained comparable basal calcium and energy levels. Parkinson's disease cybrids recovered from a carbachol-induced increase in cytosolic calcium 53% more slowly than controls even with lanthanum and thapsigargin blockade. Inhibition of complex I with the Parkinson's disease-inducing metabolite 1-methyl-4-phenylpyridinium (MPP+) similarly reduced the rate of recovery after carbachol. This MPP(+)-induced reduction in recovery rates was much more pronounced in control cybrids than in Parkinson's disease cybrids. Parkinson's disease cybrids had less carbonyl cyanide m-chlorophenylhydrazone-releasable calcium. Bypassing complex I with succinate partially restored Parkinson's disease cybrid, and MPP+ suppressed control cybrid recovery rates. The subtle alteration in calcium homeostasis of Parkinson's disease cybrids may reflect an increased susceptibility to cell death under circumstances not ordinarily toxic.

Further evidence for mitochondrial dysfunction in progressive supranuclear palsy.

Recent data from our laboratory have identified a role for mitochondrial dysfunction in the pathogenesis of progressive supranuclear palsy (PSP). To extend this finding, we measured key parameters of mitochondrial function in platelet-derived cytoplasmic hybrid (cybrid) cell lines expressing mitochondrial genes from patients with PSP. We observed significant decreases in aconitase activity, cellular ATP levels, and oxygen consumption in PSP cybrids as compared to control cybrids, further suggesting a contributory role of impaired mitochondrial energy metabolism in PSP, possibly due to genetic abnormalities of mitochondrial DNA.

Cyclosporin A increases resting mitochondrial membrane potential in SY5Y cells and reverses the depressed mitochondrial membrane potential of Alzheimer's disease cybrids.

Alzheimer's disease (AD) brains exhibit oxidative stress and a biochemical defect of complex IV (cytochrome oxidase, COX) of the mitochondrial electron transport chain (ETC). This defect can be transferred through mitochondrial DNA (mtDNA) into clonal SY5Y cells depleted of their mtDNA. The resulting cytoplasmic hybrids or "cybrids" retain the complex IV defect and exhibit oxidative stress. We measured the mitochondrial membrane potential (delta psi m) in AD and control cybrids via H3-tetraphenylphosphonium ion (H3-TPP+) accumulation. AD cybrids exhibited a significant (about 30%) decrease in H3-TPP+ accumulation relative to controls. Acute treatment of normal SY5Ys with azide, a COX inhibitor, moderately decreased H3-TPP+ retention and strongly inhibited COX activity in a dose-dependent manner. As the mitochondrial transition pore (MTP) can be activated by reactive oxygen species and ETC inhibitors, and its opening causes delta psi m dissipation, we tested the effects of the MTP inhibitor cyclosporin A (CsA) on TPP+ accumulation. 5mM CsA increased basal H3-TPP+ accumulation in SY5Y cells about 10-fold, corresponding to about a 2-fold increase in delta psi m. In the AD cybrids, CsA increased the apparent delta psi m to the same final levels as it did in controls. These results indicate that low-conductance MTP activity contributes significantly to resting delta psi m in SY5Y cells. We propose the novel hypothesis that the COX defect and resulting oxidative stress in AD may pathologically activate the MTP, resulting in lower delta psi m and the release of mitochondrial factors involved in apoptosis.

Calcium homeostasis and reactive oxygen species production in cells transformed by mitochondria from individuals with sporadic Alzheimer's disease.

Alzheimer's disease (AD) is associated with defects in mitochondrial function. Mitochondrial-based disturbances in calcium homeostasis, reactive oxygen species (ROS) generation, and amyloid metabolism have been implicated in the pathophysiology of sporadic AD. The cellular consequences of mitochondrial dysfunction, however, are not known. To examine these consequences, mitochondrially transformed cells (cybrids) were created from AD patients or disease-free controls. Mitochondria from platelets were fused to rho0 cells created by depleting the human neuroblastoma line SH-SY5Y of its mitochondrial DNA (mtDNA). AD cybrids demonstrated a 52% decrease in electron transport chain (ETC) complex IV activity but no difference in complex I activity compared with control cybrids or SH-SY5Y cells. This mitochondrial dysfunction suggests a transferable mtDNA defect associated with AD. ROS generation was elevated in the AD cybrids. AD cybrids also displayed an increased basal cytosolic calcium concentration and enhanced sensitivity to inositol-1,4, 5-triphosphate (InsP3)-mediated release. Furthermore, they recovered more slowly from an elevation in cytosolic calcium induced by the InsP3 agonist carbachol. Mitochondrial calcium buffering plays a major role after this type of perturbation. beta-amyloid (25-35) peptide delayed the initiation of calcium recovery to a carbachol challenge and slowed the recovery rate. Nerve growth factor reduced the carbachol-induced maximum and moderated the recovery kinetics. Succinate increased ETC activity and partially restored the AD cybrid recovery rate. These subtle alterations in calcium homeostasis and ROS generation might lead to increased susceptibility to cell death under circumstances not ordinarily toxic.

Interaction among mitochondria, mitogen-activated protein kinases, and nuclear factor-kappaB in cellular models of Parkinson's disease.

Oxidative stress induced by acute complex I inhibition with 1-methyl-4-phenylpyridinium ion activated biphasically the stress-activated c-Jun N-terminal kinase (JNK) and the early transcription factor nuclear factor-kappaB (NF-kappaB) in SH-SY5Y neuroblastoma cells. Early JNK activation was dependent on mitochondrial adenine nucleotide translocator (ANT) activity, whereas late-phase JNK activation and the cleavage of signaling proteins Raf-1 and mitogen-activated protein kinase (MAPK) kinase (MEK) kinase (MEKK)-1 appeared to be ANT-independent. Early NF-kappaB activation depended on MEK, later activation required an intact electron transport chain (ETC), and Parkinson's disease (PD) cybrid (mitochondrial transgenic cytoplasmic hybrid) cells had increased basal NF-kappaB activation. Mitochondria appear capable of signaling ETC impairment through MAPK modules and inducing protective NF-kappaB responses, which are increased by PD mitochondrial genes amplified in cybrid cells. Irreversible commitment to apoptosis in this cell model may derive from loss of Raf-1 and cleavage/activation of MEKK-1, processes reported in other models to be caspase-mediated. Therapeutic strategies that reduce mitochondrial activation of proapoptotic MAPK modules, i.e., JNK, and enhance survival pathways, i.e., NF-kappaB, may offer neuroprotection in this debilitating disease.