Shaharyar M. Khan

The Alzheimer's Disease Mitochondrial Cascade Hypothesis

We first proposed the mitochondrial cascade hypothesis of sporadic Alzheimers disease (AD) in 2004. Our core assumptions were a persons genes determine baseline mitochondrial function and durability, this durability determines how mitochondria change with advancing age, and critical changes in mitochondrial function initiate other pathologies characteristic of AD. Since then several lines of investigation report data consistent with or supportive of our hypothesis. In particular, AD endophenotype studies suggest a strong maternal genetic contribution, and links between mitochondrial function, tau phosphorylation, and amyloid-beta (Abeta) amyloidosis are increasingly recognized. As predicted, AD therapies designed to reduce Abeta thus far have had at best very limited clinical benefits; our hypothesis identifies alternative therapeutic targets. While placing mitochondria at the apex of an AD cascade certainly remains controversial, it is increasingly accepted by the AD research community that mitochondria play an important role in the late-onset forms of the disease. Even if the mitochondrial cascade hypothesis proves incorrect, considering its assumptions could potentially advance our understanding of sporadic, late-onset AD.

The Alzheimer's disease mitochondrial cascade hypothesis: Progress and perspectives

Ten years ago we first proposed the Alzheimers disease (AD) mitochondrial cascade hypothesis. This hypothesis maintains that gene inheritance defines an individuals baseline mitochondrial function; inherited and environmental factors determine rates at which mitochondrial function changes over time; and baseline mitochondrial function and mitochondrial change rates influence AD chronology. Our hypothesis unequivocally states in sporadic, late-onset AD, mitochondrial function affects amyloid precursor protein (APP) expression, APP processing, or beta amyloid (Aβ) accumulation and argues if an amyloid cascade truly exists, mitochondrial function triggers it. We now review the state of the mitochondrial cascade hypothesis, and discuss it in the context of recent AD biomarker studies, diagnostic criteria, and clinical trials. Our hypothesis predicts that biomarker changes reflect brain aging, new AD definitions clinically stage brain aging, and removing brain Aβ at any point will marginally impact cognitive trajectories. Our hypothesis, therefore, offers unique perspective into what sporadic, late-onset AD is and how to best treat it.

The Alzheimer's disease mitochondrial cascade hypothesis: An update

In 2004 we proposed the mitochondrial cascade hypothesis of sporadic Alzheimers disease (AD). Our hypothesis assumed sporadic and autosomal dominant AD are not etiologically homogeneous, considered evidence that AD pathology is not brain-limited, and incorporated aging theory. The mitochondrial cascade hypothesis asserted: (1) inheritance determines mitochondrial baseline function and durability; (2) mitochondrial durability influences how mitochondria change with age; and (3) when mitochondrial change reaches a threshold, AD histopathology and symptoms ensue. We now review the reasoning used to formulate the hypothesis, discuss pertinent interim data, and update its tenants. Readers are invited to consider the conceptual strengths and weaknesses of this hypothesis.

Oxidative Stress, Mitochondrial Dysfunction, and Stress Signaling in Alzheimers Disease

Although oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative diseases such as Alzheimers disease (AD), it remains unclear how mitochondrial oxidative stress may induce neuronal death. In a variety of tissues, cumulative oxidative stress, disrupted mitochondrial respiration, and mitochondrial damage are associated with, and may indeed promote cell death and degeneration. In this review, we examine current evidence supporting the involvement of mitochondria and mitochondrially generated stress signaling in AD and discuss potential implications for the mechanism of pathogenesis of this disease. Mitochondria are pivotal in controlling cell life and death not only by producing ATP, and sequestering calcium, but by also generating free radicals and serving as repositories for proteins which regulate the intrinsic apoptotic pathway. Perturbations in the physiological function of mitochondria inevitably disturb cell function, sensitize cells to neurotoxic insults and may initiate cell death, all significant phenomena in the pathogenesis of a number of neurodegenerative disorders including AD.

Inhibition by R(+) or S(–) pramipexole of caspase activation and cell death induced by methylpyridinium ion or beta amyloid peptide in SH‐SY5Y neuroblastoma

Cell models of neurodegenerative diseases (NDD) can involve expression of mutant nuclear genes associated with Mendelian forms of the diseases or effects of toxins believed to replicate essential disease features. Death produced by exposing neural cells to methylpyridinium ion (MPP+) or neurotoxic beta amyloid (BA) peptides is frequently used to study features of the sporadic, most prevalent forms of Parkinsons disease (PD) and Alzheimers disease (AD), respectively. We examined in replicating SH‐SY5Y human neuroblastoma cells the release of cytochrome C into cytoplasm, activation of caspases 9 and 3, and loss of calcein retention as markers of the “mitochondrial” pathway of cell death. Exposure to 5 mM MPP+, which induces apoptotic cell death within 18–24 hr, released cytochrome C within 4 hr, activated caspases 9 and 3, and reduced calcein accumulation. BA 25–35 peptide produced more rapid and greater elevations of caspase 3 activity; no effects were observed with the nontoxic BA 35–25 reverse sequence. The dependence on mitochondrial transition pore (MTP) activity of MPP+‐induced caspase activations was demonstrated by preincubation with bongkreckic acid, which blocked elevations of caspases 9 and 3. Stereoisomers of pramipexole (PPX), a free radical scavenger and inhibitor of MTP opening, inhibited caspase activation (MPP+ and BA) and restored calcein accumulation (MPP+). Our results demonstrate that MPP+ and BA can induce cell death through MTP‐dependent activation of caspase cascades. PPX stereoisomers interfere with activation of these cell death pathways and may be useful clinically as neuroprotectants in PD and AD and related diseases.

Recombinant Human Mitochondrial Transcription Factor A Stimulates Mitochondrial Biogenesis and ATP Synthesis, Improves Motor Function after MPTP, Reduces Oxidative Stress and Increases Survival after Endotoxin

Recombinant human mitochondrial transcription factor A protein (rhTFAM) was evaluated for its acute effects on cultured cells and chronic effects in mice. Fibroblasts incubated with rhTFAM acutely increased respiration in a chloramphenicol-sensitive manner. SH-SY5Y cells showed rhTFAM concentration-dependent reduction of methylpyridinium (MPP(+))-induced oxidative stress and increases in lowered ATP levels and viability. Mice treated with weekly i.v. rhTFAM showed increased mitochondrial gene copy number, complex I protein levels and ATP production rates; oxidative damage to proteins was decreased ~50%. rhTFAM treatment improved motor recovery rate after treatment with MPTP and dose-dependently improved survival in the lipopolysaccharide model of endotoxin sepsis.

Effects of single and multiple treatments with L-dihydroxyphenylalanine (L-DOPA) on dopamine receptor-G protein interactions and supersensitive immediate early gene responses in striata of rats after reserpine treatment or with unilateral nigrostriatal lesions.

We studied effects of l‐dihydroxyphenylalanine (L‐DOPA) treatment in rats following reserpine treatment or unilateral 6‐hydroxydopamine (6‐OHDA) injections into medial forebrain bundle. Quantitative in situ hybridization for mRNAs coding for the zinc finger immediate early gene (IEG) zif/268 or Jun family IEG jun b revealed that single L‐DOPA injections accentuated IEG expression 3‐ to 7‐fold in the dopamine (DA)‐depleted striatum. This increased IEG response did not derive from any alterations in DA receptor–G protein coupling, assayed by DA stimulation of 35S‐guanosine‐5′ (γ‐thio) triphosphate (35S‐GTP‐γ‐S) binding to striatal sections. Reserpine treatment increased both basal and maximal striatal DA‐stimulated 35S‐GTP‐γ‐S binding. The augmented IEG responses to single L‐DOPA treatments involved dependency on both D1 and D2 receptors and acutely to N‐methyl‐d‐aspartate (NMDA) channels. Repetitive L‐DOPA treatments yielded persistently elevated (zif/268) or additionally up‐regulated (jun b) IEG response in the denervated striatum and down‐regulated IEG responses in the control striatum. Degraded L‐DOPA responses and appearance of involuntary movements after chronic L‐DOPA use in advanced Parkinsons disease may derive from these IEG changes. J. Neurosci. Res. 55:71–79, 1999. 

Mitochondrial toxins in models of neurodegenerative diseases. II: elevated zif268 transcription and independent temporal regulation of striatal D1 and D2 receptor mRNAs and D1 and D2 receptor-binding sites in C57BL/6 mice during MPTP treatment

Sporadic Parkinsons disease (PD) may arise from a defect in complex I of the mitochondrial electron transport chain (ETC), transmitted through mitochondrial DNA mutations. The N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of experimental PD is believed to arise from loss of complex I activity in dopamine (DA) neurons after accumulation of MPP+, a potent complex I inhibitor and the two electron monoamine oxidase B oxidation product of MPTP. Acute MPP+ infusion into striatum, possibly mimicking the in vivo situation after MPTP treatment, increases release of DA and production of hydroxyl radical (-OH). We treated C57BL/6 mice with MPTP and followed the expression of the immediate-early gene zif268 in striatum as a marker of DA synaptic activity, determined the pharmacology of its activation during MPTP toxicity, and assayed the time course of MPTP effects on striatal DA transporter (DAT), and D1 and D2 DA receptor-binding sites and their mRNAs. MPTP (24 mg/kg b.i.d. for 4 doses) increased striatal zif268 expression, with peak effects observed 24 h after starting MPTP. Increased striatal zif268 was dependent mainly on DA D1 and to a lesser extent on non-NMDA glutamate receptors and was not altered by inhibition of nitric oxide synthase (NOS). Our MPTP schedule resulted in a loss of about one-third of nigral DA neurons. We observed with [3H]mazindol autoradiography that loss of striatal DAT sites after starting MPTP was heterogenous and greatest in centromedial striatum, reached a maximum at 48 h and showed a slight recovery at 2 weeks. Striatal D1 and D2 receptor-binding sites (measured with [3H]SCH23390 and [3H]spiperone binding, respectively) and mRNA levels for D1 and D2 receptors (determined with quantitative in situ hybridization) were altered after MPTP treatment in temporally independent manners. MPTP toxicity to the nigrostriatal system likely induces substantial striatal DA release in vivo and stimulates transcription of at least one major IEG, zif268, in striatal neurons. Increased striatal zif268 expression after MPTP appears to derive mainly from DA released onto D1 receptors, not by a NO-dependent process which has been described in striatal neurons in vitro. The rapid loss of striatal DA terminals after MPTP treatment alters D1 and D2 receptor sites independently of changes in their mRNA levels. Increased D1 and D2 gene transcription in this model may depend on re-innervation by DA terminals of striatal neurons and likely is not related to the increased zif268 transcription observed after MPTP.

RNA-seq analyses reveal that cervical spinal cords and anterior motor neurons from amyotrophic lateral sclerosis subjects show reduced expression of mitochondrial DNA-encoded respiratory genes, and rhTFAM may correct this respiratory deficiency

Amyotrophic lateral sclerosis (ALS) is a generally fatal neurodegenerative disease of adults that produces weakness and atrophy due to dysfunction and death of upper and lower motor neurons. We used RNA-sequencing (RNA-seq) to analyze expression of all mitochondrial DNA (mtDNA)-encoded respiratory genes in ALS and CTL human cervical spinal cords (hCSC) and isolated motor neurons. We analyzed with RNA-seq mtDNA gene expression in human neural stem cells (hNSC) exposed to recombinant human mitochondrial transcription factor A (rhTFAM), visualized in 3-dimensions clustered gene networks activated by rhTFAM, quantitated their interactions with other genes and determined their gene ontology (GO) families. RNA-seq and quantitative PCR (qPCR) analyses showed reduced mitochondrial gene expression in ALS hCSC and ALS motor neurons isolated by laser capture microdissection (LCM), and revealed that hNSC and CTL human cervical spinal cords were similar. Rats treated with i.v. rhTFAM showed a dose-response increase in brain respiration and an increase in spinal cord mitochondrial gene expression. Treatment of hNSC with rhTFAM increased expression of mtDNA-encoded respiratory genes and produced one major and several minor clusters of gene interactions. Gene ontology (GO) analysis of rhTFAM-stimulated gene clusters revealed enrichment in GO families involved in RNA and mRNA metabolism, suggesting mitochondrial-nuclear signaling. In postmortem ALS hCSC and LCM-isolated motor neurons we found reduced expression of mtDNA respiratory genes. In hNSCs rhTFAM increased mtDNA gene expression and stimulated mRNA metabolism by unclear mechanisms. rhTFAM may be useful in improving bioenergetic function in ALS.