Amyotrophic lateral sclerosis (ALS) is the third most common adult-onset neurodegenerative disease. It causes the degeneration of motoneurons and is fatal due to paralysis, particularly of respiratory muscles. ALS can be inherited, and specific disease-causing genes have been identified, but the mechanisms causing motoneuron death in ALS are not understood. No effective treatments exist for ALS. One well-studied theory of ALS pathogenesis involves faulty RNA editing and abnormal activation of specific glutamate receptors as well as failure of glutamate transport resulting in glutamate excitotoxicity; however, the excitotoxicity theory is challenged by the inability of anti-glutamate drugs to have major disease-modifying effects clinically. Nevertheless, hyperexcitability of upper and lower motoneurons is a feature of human ALS and transgenic (tg) mouse models of ALS. Motoneuron excitability is strongly modulated by synaptic inhibition mediated by presynaptic glycinergic and GABAergic innervations and postsynaptic glycine receptors (GlyR) and GABAA receptors; yet, the integrity of inhibitory systems regulating motoneurons has been understudied in experimental models, despite findings in human ALS suggesting that they may be affected. We have found in tg mice expressing a mutant form of human superoxide dismutase-1 (hSOD1) with a Gly93 → Ala substitution (G93A-hSOD1), causing familial ALS, that subsets of spinal interneurons degenerate. Inhibitory glycinergic innervation of spinal motoneurons becomes deficient before motoneuron degeneration is evident in G93A-hSOD1 mice. Motoneurons in these ALS mice also have insufficient synaptic inhibition as reflected by smaller GlyR currents, smaller GlyR clusters on their plasma membrane, and lower expression of GlyR1α mRNA compared to wild-type motoneurons. In contrast, GABAergic innervation of ALS mouse motoneurons and GABAA receptor function appear normal. Abnormal synaptic inhibition resulting from dysfunction of interneurons and motoneuron GlyRs is a new direction for unveiling mechanisms of ALS pathogenesis that could be relevant to new therapies for ALS.

Researchers used transgenic mice expressing enhanced-green fluorescent protein (eGFP) driven by either the glycine transporter-2 gene promoter to specifically visualize glycinergic interneurons or the homeobox-9 (Hb9) gene promoter to visualize motoneurons for assessing their vulnerabilities to excitotoxins in vivo. Stereotaxic excitotoxic lesions were made in adult male and female mouse lumbar spinal cord with the specific N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA) and the non-NMDA ion channel glutamate receptor agonist kainic acid (KA). QA and KA induced large-scale degeneration of glycinergic interneurons in spinal cord. Glycinergic interneurons were more sensitive than motoneurons to NMDA receptor-mediated and non-NMDA glutamate receptor-mediated excitotoxicity. Outcome after spinal cord excitotoxicity was gender-dependent, with males showing greater sensitivity than females. Excitotoxic degeneration of spinal interneurons resembled apoptosis, while motoneuron degeneration appeared non-apoptotic. Perikaryal mitochondrial accumulation was antecedent to both NMDA and non-NMDA receptor-mediated excitotoxic stimulation of interneurons and motoneurons. Genetic ablation of cyclophilin D, a regulator of the mitochondrial permeability transition pore (mPTP), protected both interneurons and motoneurons from excitotoxicity. The results demonstrate in adult mouse spinal cord that glycinergic interneurons are more sensitive than motoneurons to excitotoxicity that stimulates mitochondrial accumulation, and that the mPTP has pro-death functions mediating apoptotic and non-apoptotic neuronal degeneration in vivo.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease that causes degeneration of motor neurons and paralysis. Approximately 20% of familial ALS cases have been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene but it is unclear how mutations in the protein result in motor neuron degeneration. Transgenic (tg) mice expressing mutated forms of human SOD1 (hSOD1) develop clinical and pathological features similar to those of ALS. We used tg mice expressing hSOD1-G93A, hSOD1-G37R, and hSOD1-wild type to investigate a new subcellular pathology involving mutant hSOD1 protein prominently localizing to the nuclear compartment and disruption of the architecture of nuclear gems. We developed methods for extracting relatively pure cell nucleus fractions from mouse CNS tissues and demonstrate low nuclear presence of endogenous SOD1 in mouse brain and spinal cord, but prominent nuclear accumulation of hSOD1-G93A, -G37R and -wild type in tg mice. hSOD1 concentrated in nuclei of spinal cord cells, particularly motor neurons, at a young age. The survival motor neuron protein (SMN) complex is disrupted in motor neuron nuclei prior to disease onset in hSOD1-G93A and -G37R mice; age-matched hSOD1-wild type mice did not show SMN disruption despite a nuclear presence. Our data suggest new mechanisms involving hSOD1 accumulation in the cell nucleus and mutant hSOD1-specific perturbations in SMN localization with disruption of the nuclear SMN complex in the ALS model mice and suggest overlap of pathogenic mechanisms with spinal muscular atrophy.

Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) are the most common human adult-onset neurodegenerative diseases. They are characterized by prominent age-related neurodegeneration in selectively vulnerable neural systems. Some forms of AD, PD, and ALS are inherited, and genes causing these diseases have been identified. Nevertheless, the mechanisms of the neuronal degeneration in these familial diseases, and in the more common idiopathic (sporadic) diseases, are unresolved. Genetic, biochemical, and morphological analyses of human AD, PD, and ALS, as well as their cell and animal models, reveal that mitochondria could have roles in this neurodegeneration. The varied functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and the overlying genetic variations. In AD, alterations in enzymes involved in oxidative phosphorylation, oxidative damage, and mitochondrial binding of Aβ and amyloid precursor protein have been reported. In PD, mutations in mitochondrial proteins have been identified and mitochondrial DNA mutations have been found in neurons in the substantia nigra. In ALS, changes occur in mitochondrial respiratory chain enzymes and mitochondrial programmed cell death proteins. Transgenic mouse models of human neurodegenerative disease are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria and the mitochondrial permeability transition pore. This chapter reviews several aspects of mitochondrial biology and how mitochondrial pathobiology might contribute to the mechanisms of neurodegeneration in AD, PD, and ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motoneurons. Recently we studied glycine receptors (GlyRs) in motoneurons in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). Living motoneurons in dissociated spinal cord cultures were identified by using transgenic mice expressing eGFP driven by the Hb9 promoter. We showed that GlyR-mediated currents were reduced in large-sized (diameter >28 µm) Hb9-eGFP+ motoneurons from G93A-SOD1 embryonic mice. Here we analyze GlyR currents in a morphologically distinct subgroup of medium-sized (diameter 10–28 µm) Hb9-eGFP+ motoneurons, presumably gamma or slow-type alpha motoneurons. We find that glycine-induced current densities were not altered in medium-sized G93A-SOD1 motoneurons. No significant differences in glycinergic mIPSCs were observed between G93A-SOD1 and control medium-sized motoneurons. These results indicate that GlyR deficiency early in the disease process of ALS is specific for large alpha motoneurons.

Striatal neurons are highly vulnerable to hypoxia-ischemia (HI) in term newborns. In a piglet model of HI, striatal neurons develop oxidative stress and organelle disruption by 3–6 h of recovery and ischemic cytopathology over 6–24 h of recovery. We tested the hypothesis that early treatment with the antioxidants EUK-134 (a manganese-salen derivative that acts as a scavenger of superoxide, hydrogen peroxide, nitric oxide or NO and peroxynitrite) or edaravone (MCI-186, a scavenger of hydroxyl radical and NO) protects striatal neurons from HI. Anesthetized newborn piglets were subjected to 40 min of hypoxia and 7 min of airway occlusion. At 30 min after resuscitation, the piglets received vehicle, EUK-134 or edaravone. Drug treatment did not affect arterial blood pressure, blood gases, blood glucose or rectal temperature. At 4 days of recovery, the density of viable neurons in the putamen of vehicle-treated piglets was 12 ± 6% (±SD) of sham-operated control density. Treatment with EUK-134 increased viability to 41 ± 17%, and treatment with edaravone increased viability to 39 ± 19%. In the caudate nucleus, neuronal viability was increased from 54 ± 11% in the vehicle group to 78 ± 15% in the EUK-134 group and to 73 ± 13% in the edaravone group. Antioxidant drug treatment accelerated recovery from neurologic deficits and decreased oxidative and nitrative damage to nucleic acids. Treatment with EUK-134 reduced the HI-induced formation of protein carbonyl groups and tyrosine nitration at 3 h of recovery. We conclude that systemic administration of antioxidant agents by 30 min after resuscitation from HI can reduce oxidative stress and salvage neurons in the highly vulnerable striatum in a large-animal model of neonatal HI. Therefore, oxidative stress is an important mechanism for this injury, and antioxidant therapy is a rational, mechanism-based approach to neuroprotection in the newborn brain.

Na+, Ca2+- permeable acid-sensing ion channel 1a (ASIC1a) is involved in the pathophysiologic process of adult focal brain ischemia. However, little is known about its role in the pathogenesis of global cerebral ischemia or newborn hypoxia-ischemia (H-I). Here, using a newborn piglet model of asphyxia-induced cardiac arrest, we investigated the effect of ASIC1a-specific blocker psalmotoxin-1 on neuronal injury. During asphyxia and the first 30 mins of recovery, brain tissue pH fell below 7.0, the approximate activation pH of ASIC1a. Psalmotoxin-1 injection at 20 mins before hypoxia, but not at 20 mins of recovery, partially protected the striatonigral and striatopallidal neurons in putamen. Psalmotoxin-1 pretreatment largely attenuated the increased protein kinase A-dependent phosphorylation of DARPP-32 and N-methyl-D-aspartate (NMDA) receptor NR1 subunit and decreased nitrative and oxidative damage to proteins at 3 h of recovery. Pretreatment with NMDA receptor antagonist MK-801 also provided partial neuroprotection in putamen, and combined pretreatment with psalmotoxin-1 and MK-801 yielded additive neuroprotection. These results indicate that ASIC1a activation contributes to neuronal death in newborn putamen after H-I through mechanisms that may involve protein kinase A-dependent phosphorylation of NMDA receptor and nitrative and oxidative stress.

Amyotrophic lateral sclerosis (ALS) is a rapidly evolving and fatal adult-onset neurological disease characterized by progressive degeneration of motoneurons. Our previous study showed that glycinergic innervation of spinal motoneurons is deficient in an ALS mouse model expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). In this study we have examined, using whole-cell patch clamp recordings, glycine receptor (GlyR)-mediated currents in spinal motoneurons from these transgenic mice. We developed a dissociated spinal cord culture model using embryonic transgenic mice expressing eGFP driven by the Hb9 promoter. Motoneurons were identified as Hb9-eGFP+ neurons with a characteristic morphology. To examine GlyRs in ALS motoneurons, we bred G93A-SOD1 mice to Hb9-eGFP mice and compared glycine-evoked currents in cultured Hb9-eGFP+ motoneurons prepared from G93A-SOD1 embryos and from their non-transgenic littermates. Glycine-evoked current density was significantly smaller in the G93A-SOD1 motoneurons compared with control. Furthermore, the averaged current densities of spontaneous glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were significantly smaller in the G93A-SOD1 motoneurons than in control motoneurons. No significant differences in GABA-induced currents and GABAergic mIPSCs were observed between G93A-SOD1 and control motoneurons. Quantitative single-cell RT-PCR found lower GlyRα1 subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the reduction of GlyR current may result from the downregulation of GlyR mRNA expression in motoneurons. Immunocytochemistry demonstrated a decrease of surface postsynaptic GlyR on G93A-SOD1 motoneurons. Our study suggests that selective alterations in GlyR function contribute to inhibitory insufficiency in motoneurons early in the disease process of ALS.

DNA methylation is an epigenetic mechanism for gene silencing engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to cytosine residues in gene regulatory regions. It is unknown if aberrant DNA methylation can cause neurodegeneration. We tested the hypothesis that Dnmts can mediate neuronal cell death. Enforced expression of Dnmt3a induced degeneration of cultured NSC34 cells. During apoptosis of NSC34 cells induced by camptothecin, levels of Dnmt1 and Dnmt3a increased five-fold and two-fold, respectively, and 5-methylcytosine accumulated in nuclei. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocked apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with RG108 and procainamide protected cultured neurons from excessive DNA methylation and apoptosis. In vivo, Dnmt1 and Dnmt3a are expressed differentially during mouse brain and spinal cord maturation and in adulthood when Dnmt3a is abundant in synapses and mitochondria. Dnmt1 and Dnmt3a are expressed in motor neurons of adult mouse spinal cord, and, during their apoptosis induced by sciatic nerve avulsion, nuclear and cytoplasmic 5-methylcytosine immunoreactivity, Dnmt3a protein levels, and Dnmt enzyme activity increased preapoptotically. Inhibition of Dnmts with RG108 blocked completely the increase in 5-methycytosine and the apoptosis of motor neurons in mice. In human amyotrophic lateral sclerosis (ALS), motor neurons showed changes in Dnmt1, Dnmt3a, and 5-methylcytosine similar to experimental models. Thus, motor neurons can engage epigenetic mechanisms to drive apoptosis, involving Dnmt upregulation and increased DNA methylation. These cellular mechanisms could be relevant to human ALS pathobiology and disease treatment.

Despite the introduction of therapeutic hypothermia, neonatal hypoxic ischemic (HI) brain injury remains a common cause of developmental disability. Development of rational adjuvant therapies to hypothermia requires understanding of the pathways of cell death and survival modulated by HI. The conceptualization of the apoptosis-necrosis “continuum” in neonatal brain injury predicts mechanistic interactions between cell death and hydrid forms of cell death such as programmed or regulated necrosis. Many of the components of the signaling pathway regulating programmed necrosis have been studied previously in models of neonatal HI. In some of these investigations, they participate as part of the apoptotic pathways demonstrating clear overlap of programmed death pathways. Receptor interacting protein (RIP)-1 is at the crossroads between types of cellular death and survival and RIP-1 kinase activity triggers formation of the necrosome (in complex with RIP-3) leading to programmed necrosis. Neuroprotection afforded by the blockade of RIP-1 kinase following neonatal HI suggests a role for programmed necrosis in the HI injury to the developing brain. Here, we briefly review the state of the knowledge about the mechanisms behind programmed necrosis in neonatal brain injury recognizing that a significant proportion of these data derive from experiments in cultured cell and some from in vivo adult animal models. There are still more questions than answers, yet the fascinating new perspectives provided by the understanding of programmed necrosis in the developing brain may lay the foundation for new therapies for neonatal HI.

Effective therapies are needed for the treatment of amyotrophic lateral sclerosis (ALS), a fatal type of motor neuron disease. Morphological, biochemical, molecular genetic, and cell/animal model studies suggest that mitochondria have potentially diverse roles in neurodegenerative disease mechanisms and neuronal cell death. In human ALS, abnormalities have been found in mitochondrial structure, mitochondrial respiratory chain enzymes, and mitochondrial cell death proteins indicative of some non-classical form of programmed cell death. Mouse models of ALS are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria. This minireview summarizes work on the how malfunctioning mitochondria might contribute to neuronal death in ALS through the biophysical entity called the mitochondrial permeability pore (mPTP). The major protein components of the mPTP are enriched in mouse motor neurons. Early in the course of disease in ALS mice expressing human mutant superoxide dismutase-1, mitochondria in motor neurons undergo trafficking abnormalities and dramatic remodeling resulting in the formation of mega-mitochondria and coinciding with increased protein carbonyl formation and nitration of mPTP components. The genetic deletion of a major mPTP component, cyclophilin D, has robust effects in ALS mice by delaying disease onset and extending survival. Thus, attention should be directed to the mPTP as rational target for the development of drugs designed to treat ALS.

Cerebral cortical neuron degeneration occurs in brain disorders manifesting throughout life, but the mechanisms are understood poorly. We used cultured embryonic mouse cortical neurons and an in vivo mouse model to study mechanisms of DNA damaged-induced apoptosis in immature and differentiated neurons. p53 drives apoptosis of immature and differentiated cortical neurons through its rapid and prominent activation stimulated by DNA strand breaks induced by topoisomerase-I and -II inhibition. Blocking p53-DNA transactivation with α-pifithrin protects immature neurons; blocking p53-mitochondrial functions with μ-pifithrin protects differentiated neurons. Mitochondrial death proteins are upregulated in apoptotic immature and differentiated neurons and have nonredundant proapoptotic functions; Bak is more dominant than Bax in differentiated neurons. p53 phosphorylation is mediated by ataxia telangiectasia mutated (ATM) kinase. ATM inactivation is antiapoptotic, particularly in differentiated neurons, whereas inhibition of c-Abl protects immature neurons but not differentiated neurons. Cell death protein expression patterns in mouse forebrain are mostly similar to cultured neurons. DNA damage induces prominent p53 activation and apoptosis in cerebral cortex in vivo. Thus, DNA strand breaks in cortical neurons induce rapid p53-mediated apoptosis through actions of upstream ATM and c-Abl kinases and downstream mitochondrial death proteins. This molecular network operates through variations depending on neuron maturity.

Necrostatin-1 inhibits receptor-interacting protein (RIP)-1 kinase and programmed necrosis and is neuroprotective in adult rodent models. Owing to the prominence of necrosis and continuum cell death in neonatal hypoxia–ischemia (HI), we tested whether necrostatin was neuroprotective in the developing brain. Postnatal day (P)7 mice were exposed to HI and injected intracerebroventricularly with 0.1 μL of 80 μmol necrostatin, Nec-1, 5-(1H-Indol-3-ylmethyl)-(2-thio-3-methyl) hydantoin, or vehicle. Necrostatin significantly decreased injury in the forebrain and thalamus at P11 and P28. There was specific neuroprotection in necrostatin-treated males. Necrostatin treatment decreased necrotic cell death and increased apoptotic cell death. Hypoxia–ischemia enforced RIP1–RIP3 complex formation and inhibited RIP3–FADD (Fas-associated protein with death domain) interaction, and these effects were blocked by necrostatin. Necrostatin also decreased HI-induced oxidative damage to proteins and attenuated markers of inflammation coincidental with decreased nuclear factor-κB and caspase 1 activation, and FLIP ((Fas-associated death-domain-like IL-1β-converting enzyme)-inhibitory protein) gene and protein expression. In this model of severe neonatal brain injury, we find that cellular necrosis can be managed therapeutically by a single dose of necrostatin, administered after HI, possibly by interrupting RIP1–RIP3-driven oxidative injury and inflammation. The effects of necrostatin treatment after HI reflect the importance of necrosis in the delayed phases of neonatal brain injury and represent a new direction for therapy of neonatal HI.

Background

Mitochondria have roles or appear to have roles in the pathogenesis of several chronic age-related and acute neurological disorders, including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, Parkinson's disease, and cerebral ischemia, and could be critical targets for development of rational mechanism-based, disease-modifying therapeutics for treating these disorders effectively. A deeper understanding of neural tissue mitochondria pathobiologies as definitive mediators of neural injury, disease, and cell death merits further study, and the development of additional tools to study neural mitochondria will help achieve this unmet need.

Results

We created transgenic mice that express the coral (Discosoma sp.) red fluorescent protein DsRed2 specifically in mitochondria of neurons using a construct engineered with a Thy1 promoter, specific for neuron expression, to drive expression of a fusion protein of DsRed2 with a mitochondrial targeting sequence. The biochemical and histological characterization of these mice shows the expression of mitochondrial-targeted DsRed2 to be specific for mitochondria and concentrated in distinct CNS regions, including cerebral cortex, hippocampus, thalamus, brainstem, and spinal cord. Red fluorescent mitochondria were visualized in cerebral cortical and hippocampal pyramidal neurons, ventrobasal thalamic neurons, subthalamic neurons, and spinal motor neurons. For the purpose of proof of principle application, these mice were used in excitotoxicity paradigms and double transgenic mice were generated by crossing Thy1-mitoDsRed2 mice with transgenic mice expressing enhanced-GFP (eGFP) under the control of the Hlxb9 promoter that drives eGFP expression specifically in motor neurons and by crossing Thy1-mitoDsRed2 mice to amyotrophic lateral sclerosis (ALS) mice expressing human mutant superoxide dismutase-1.

Conclusions

These novel transgenic mice will be a useful tool for better understanding the biology of mitochondria in mouse and cellular models of human neurological disorders as exemplified by the mitochondrial swelling and fission seen in excitotoxicity and mouse ALS.

DNA damage is a form of cell stress and injury that has been implicated in the pathogenesis of many neurologic disorders, including amyotrophic lateral sclerosis, Alzheimer disease, Down syndrome, Parkinson disease, cerebral ischemia, and head trauma. However, most data reveal only associations, and the role for DNA damage in direct mechanisms of neurodegeneration is vague with respect to being a definitive upstream cause of neuron cell death, rather than a consequence of the degeneration. Although neurons seem inclined to develop DNA damage during oxidative stress, most of the existing work on DNA damage and repair mechanisms has been done in the context of cancer biology using cycling non-neuronal cells but not nondividing (i.e. postmitotic) neurons. Nevertheless, the identification of mutations in genes that encode proteins that function in DNA repair and DNA damage response in human hereditary DNA repair deficiency syndromes and ataxic disorders is establishing a mechanistic precedent that clearly links DNA damage and DNA repair abnormalities with progressive neurodegeneration. This review summarizes DNA damage and repair mechanisms and their potential relevance to the evolution of degeneration in postmitotic neurons.

Ablation of mouse occipital cortex induces precisely timed and uniform p53-modulated and Bax-dependent apoptosis of thalamocortical projection neurons in the dorsal lateral geniculate nucleus (LGN) by 7 days postlesion. We tested the hypothesis that this neuronal apoptosis is initiated by oxidative stress and the mitochondrial permeability transition pore (mPTP). Pre-apoptotic LGN neurons accumulate mitochondria, Zn2+ and Ca2+, and generate higher levels of reactive oxygen species (ROS), including superoxide, nitric oxide (NO) and peroxynitrite, than LGN neurons with an intact cortical target. Pre-apoptosis of LGN neurons is associated with increased formation of protein carbonyls, protein nitration, and protein S-nitrosylation. Genetic deletion of nitric oxide synthase 1 (nos1) and inhibition of NOS1 with nitroindazole protected LGN neurons from apoptosis, revealing NO as a mediator. Putative components of the mPTP are expressed in mouse LGN, including the voltage-dependent anion channel (VDAC), adenine nucleotide translocator (ANT), and cyclophilin D (CyPD). Nitration of CyPD and ANT in LGN mitochondria occurs by 2 days after cortical injury. Chemical cross-linking showed that LGN neuron pre-apoptosis is associated with formation of CyPD and VDAC oligomers, consistent with mPTP formation. Mice without CyPD are rescued from neuron apoptosis as are mice treated with the mPTP inhibitors TRO-19622 and TAT-Bcl-XL-BH4. Manipulation of the mPTP markedly attenuated the early pre-apoptotic production of reactive oxygen/nitrogen species in target-deprived neurons. Our results demonstrate in adult mouse brain neurons that the mPTP functions to enhance ROS production and the mPTP and NO trigger apoptosis; thus, the mPTP is a target for neuroprotection in vivo.

Genetic alterations in α-synuclein cause autosomal dominant familial Parkinsonism and may contribute to sporadic Parkinson's disease (PD). Synphilin-1 is an α-synuclein-interacting protein, with implications in PD pathogenesis related to protein aggregation. Currently, the in vivo role of synphilin-1 in α-synuclein-linked pathogenesis is not fully understood. Using the mouse prion protein promoter, we generated synphilin-1 transgenic mice, which did not display PD-like phenotypes. However, synphilin-1/A53T α-synuclein double-transgenic mice survived longer than A53T α-synuclein single-transgenic mice. There were attenuated A53T α-synuclein-induced motor abnormalities and decreased astroglial reaction and neuronal degeneration in brains in double-transgenic mice. Overexpression of synphilin-1 decreased caspase-3 activation, increased beclin-1 and LC3 II expression and promoted formation of aggresome-like structures, suggesting that synphilin-1 alters multiple cellular pathways to protect against neuronal degeneration. These studies demonstrate that synphilin-1 can diminish the severity of α-synucleinopathy and play a neuroprotective role against A53T α-synuclein toxicity in vivo.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes skeletal muscle paralysis. Familial forms of ALS are linked to mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human SOD1 (hSOD1) toxicity to MNs are unknown. We hypothesized that skeletal muscle is a primary site of pathogenesis in ALS that triggers MN degeneration. We created transgenic (tg) mice expressing wild-type-, G37R- and G93A-hSOD1 gene variants only in skeletal muscle. These tg mice developed age-related neurologic and pathologic phenotypes consistent with ALS. Affected mice showed limb weakness and paresis with motor deficits. Skeletal muscles developed severe pathology involving oxidative damage, protein nitration, myofiber cell death and marked neuromuscular junction (NMJ) abnormalities. Spinal MNs developed distal axonopathy and formed ubiquitinated inclusions and degenerated through an apoptotic-like pathway involving capsase-3. Mice expressing wild-type and mutant forms of hSOD1 developed MN pathology. These results demonstrate that human SOD1 in skeletal muscle has a causal role in ALS and identify a new non-autonomous mechanism for MN degeneration explaining their selective vulnerability. The discovery of instigating molecular toxicities or disease progression determinants within skeletal muscle could be very valuable for the development of new effective therapies for the treatment and cure of ALS.

Effective therapies are needed for amyotrophic lateral sclerosis (ALS), a debilitating and fatal motor neuron disease. Cell and animal models of ALS are begsinning to reveal possible principles governing the biology of motor neuron-selective vulnerability that implicate mitochondria and the mitochondrial permeability pore (mPTP). Proteins associated with the mPTP are known to be enriched in motor neurons and the genetic deletion of a major regulator of the mPTP has robust effects in ALS transgenic mice, delaying disease onset and extending survival. Thus, the mPTP is a rational, mechanism-based target for the development of drugs designed to treat ALS. Trophos SA has discovered olesoxime (TRO-19622), a small-molecule with a cholesterol-like structure, which has remarkable neuroprotective properties for motor neurons in cell culture and in rodents. Olesoxime appears to act on mitochondria, possibly at the mPTP. Phase I clinical trials of olesoxime have been completed successfully. Olesoxime is well tolerated and achieves levels predicted to be clinically effective when administered orally. It has been granted orphan drug status for the treatment of ALS in the US and for the treatment of spinal muscular atrophy in the EU. Phase II/III clinical trials are in progress in Europe.

Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) are the most common human adult-onset neurodegenerative diseases. They are characterized by prominent age-related neurodegeneration in selectively vulnerable neural systems. Some forms of AD, PD, and ALS are inherited, and genes causing these diseases have been identified. Nevertheless, the mechanisms of the neuronal cell death are unresolved. Morphological, biochemical, genetic, as well as cell and animal model studies reveal that mitochondria could have roles in this neurodegeneration. The functions and properties of mitochondria might render subsets of selectively vulnerable neurons intrinsically susceptible to cellular aging and stress and overlying genetic variations, triggering neurodegeneration according to a cell death matrix theory. In AD, alterations in enzymes involved in oxidative phosphorylation, oxidative damage, and mitochondrial binding of Aβ and amyloid precursor protein have been reported. In PD, mutations in putative mitochondrial proteins have been identified and mitochondrial DNA mutations have been found in neurons in the substantia nigra. In ALS, changes occur in mitochondrial respiratory chain enzymes and mitochondrial cell death proteins. Transgenic mouse models of human neurodegenerative disease are beginning to reveal possible principles governing the biology of selective neuronal vulnerability that implicate mitochondria and the mitochondrial permeability transition pore. This review summarizes how mitochondrial pathobiology might contribute to neuronal death in AD, PD, and ALS and could serve as a target for drug therapy.

In adult stroke models, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), a sigma receptor agonist, attenuates activity of neuronal nitric oxide synthase (nNOS), blunts ischemia-induced nitric oxide production, and provides neuroprotection. Here, we tested the hypothesis that PPBP attenuates neuronal damage in a model of global hypoxia-ischemia (H–I) in newborn piglets. Piglets subjected to hypoxia followed by asphyxic cardiac arrest were treated with saline or two dosing regimens of PPBP after resuscitation. Sigma-1 receptors were found in striatal neurons. PPBP dose-dependently protected neurons in putamen at 4 days of recovery from H–I. Immunoblots of putamen extracts at 3 h of recovery showed that PPBP decreased H–I-induced recruitment of nNOS in the membrane fraction and reduced the association of nNOS with NMDA receptor NR2 subunit. The latter effect was associated with changes in the coupling of nNOS to postsynaptic density-95 (PSD-95), but not NR2-PSD-95 interactions. Moreover, PPBP suppressed NOS activity in the membrane fraction and reduced H–I–induced nitrative and oxidative damage to proteins and nucleic acids. These findings indicate that PPBP protects striatal neurons in a large animal model of neonatal H–I and that the protection is associated with decreased coupling of nNOS to PSD-95.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs). The molecular pathogenesis of ALS is not understood, thus effective therapies for this disease are lacking. Some forms of ALS are inherited by mutations in the superoxide dismutase-1 (SOD1) gene. Transgenic mice expressing human Gly93 → Ala (G93A) mutant SOD1 (mSOD1) develop severe MN disease, oxidative and nitrative damage, and mitochondrial pathology that appears to involve nitric oxide-mediated mechanisms. We used G93A-mSOD1 mice to test the hypothesis that the degeneration of MNs is associated with an aberrant up-regulation of the inducible form of nitric oxide synthase (iNOS or NOS2) activity within MNs. Western blotting and immunoprecipitation showed that iNOS protein levels in mitochondrial-enriched membrane fractions of spinal cord are increased significantly in mSOD1 mice at pre-symptomatic stages of disease. The catalytic activity of iNOS was also increased significantly in mitochondrial-enriched membrane fractions of mSOD1 mouse spinal cord at pre-symptomatic stages of disease. Reverse transcription-PCR showed that iNOS mRNA was present in the spinal cord and brainstem MN regions in mice and was increased in pre-symptomatic and early symptomatic mice. Immunohistochemistry showed that iNOS immunoreactivty was up-regulated first in spinal cord and brainstem MNs in pre-symptomatic and early symptomatic mice and then later in the course of disease in numerous microglia and few astrocytes. iNOS accumulated in the mitochondria in mSOD1 mouse MNs. iNOS immunoreactivity was also up-regulated in Schwann cells of peripheral nerves and was enriched particularly at the paranodal regions of the nodes of Ranvier. Drug inhibitors of iNOS delayed disease onset and significantly extended the lifespan of G93A-mSOD1 mice. This work identifies two new potential early mechanisms for MN degeneration in mouse ALS involving iNOS at MN mitochondria and Schwann cells and suggests that therapies targeting iNOS might be beneficial in treating human ALS.

The Baltimore Longitudinal Study of Aging (BLSA) was established in 1958 and is one the oldest prospective studies of aging in the USA and the world. The BLSA is supported by the National Institute of Aging (NIA) and its mission is to learn what happens to people as they get old and how to sort out changes due to aging and from those due to disease or other causes. In 1986, an autopsy program combined with comprehensive neurologic and cognitive evaluations was established in collaboration with the Johns Hopkins University Alzheimer’s Disease Research Center (ADRC). Since then, 211 subjects have undergone autopsy. Here we review the key clinical neuropathological correlations from this autopsy series. The focus is on the morphological and biochemical changes that occur in normal aging, and the early neuropathological changes of neurodegenerative diseases, especially Alzheimer’s disease (AD). We highlight the combined clinical, pathologic, morphometric, and biochemical evidence of asymptomatic AD, a state characterized by normal clinical evaluations in subjects with abundant AD pathology. We conclude that in some individuals, successful cognitive aging results from compensatory mechanisms that occur at the neuronal level (i.e., neuronal hypertrophy and synaptic plasticity) whereas a failure of compensation may culminate in disease.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons (MNs) that causes paralysis. Some forms of ALS are inherited, caused by mutations in the superoxide dismutase-1 (SOD1) gene. The mechanisms of human mutant SOD1 (mSOD1) toxicity to MNs are unresolved. Mitochondria in MNs might be key sites for ALS pathogenesis, but cause-effect relationships between mSOD1 and mitochrondiopathy need further study. We used transgenic mSOD1 mice to test the hypothesis that the mitochondrial permeability transition pore (mPTP) is involved in the MN degeneration of ALS. Components of the multi-protein mPTP are expressed highly in mouse MNs, including the voltage-dependent anion channel, adenine nucleotide translocator (ANT), and cyclophilin D (CyPD), and are present in mitochondria marked by manganese SOD. MNs in pre-symptomatic mSOD1-G93A mice form swollen megamitochondria with CyPD immunoreactivity. Early disease is associated with mitochondrial cristae remodeling and matrix vesiculation in ventral horn neuron dendrites. MN cell bodies accumulate mitochondria derived from the distal axons projecting to skeletal muscle. Incipient disease in spinal cord is associated with increased oxidative and nitrative stress, indicated by protein carbonyls and nitration of CyPD and ANT. Reducing the levels of CyPD by genetic ablation significantly delays disease onset and extends the lifespan of G93A-mSOD1 mice expressing high and low levels of mutant protein in a gender-dependent pattern. These results demonstrate that mitochondria have causal roles in the disease mechanisms in MNs in ALS mice. This work defines a new mitochondrial mechanism for MN degeneration in ALS.

Objective

Clinical MR studies show delayed and ongoing neurodegeneration following neonatal hypoxia-ischemia (HI), but the mechanisms and timing of this neurodegeneration remain unclear. We use ex vivo Diffusion Tensor Imaging (DTI) and brain neuropathology to determine if selective injury to white matter tracts occurs following neonatal HI in mice resulting in neural system associated attrition in remote regions and at delayed time points.

Methods

The Rice Vannucci model (unilateral carotid ligation + 45 minutes of hypoxia FIO2=0.08) was used to cause brain injury in postnatal (p)7 C57BL6 mice and ex vivo DTI and correlative neuropathology were performed at p8,p11,p15,p21,p28, and p42.

Results

DTI provides excellent contrast visualization of unmyelinated white matter in the immature mouse brain. Acute severe ipsilateral injury to the hippocampus is seen with both histopathology and diffusion-weighted MRI 24 hours post injury. Injury to axons is evident 24 hours after HI in the hippocampal alveus. By p11 and continuing until p28, the ipsilateral fimbria fornix degenerates. Beginning at p15, there is injury and loss of axons entering the ipsilateral septal nucleus followed by ipsilateral septal atrophy. Volume loss in the hippocampus is rapid and severe but is subacute and significantly slower in the ipsilateral septum. Neonatal HIE, also interrupts the normal developmental increase in fractional anisotropy in the ipsilateral fimbria but not in the contralateral fimbria from p8 to p42.

Interpretation

In the neonatal brain there is a progressive systems-preferential injury following HI. DTI allows unparalleled visualization of this neural-network associated attrition so that it can be followed longitudinally in the developing brain.