Mechanisms Of Neuronal Degeneration Research Articles

Brain Slice Derived Nerve Fibers Grow along Microcontact Prints and are Stimulated by Beta-Amyloid(42).

Alzheimer's disease is characterized by extracellular beta-amyloid plaques, intraneuronal tau neurofibrillary tangles and excessive neurodegeneration. The mechanisms of neuron degeneration and the potential of these neurons to form new nerve fibers for compensation remain elusive. The present study aimed to evaluate the impact of beta-amyloid and tau on new formations of nerve fibers from mouse organotypic brain slices connected to collagen-based microcontact prints. Organotypic brain slices of postnatal day 8-10 wild-type mice were connected to established collagen-based microcontact prints loaded with polyornithine to enhance nerve fiber outgrowth. Human beta-amyloid(42) or P301S mutated aggregated tau was co-loaded to the prints. Nerve fibers were immunohistochemically stained with neurofilament antibodies. The physiological activity of outgrown neurites was tested with neurotracer MiniRuby, voltage-sensitive dye FluoVolt, and calcium-sensitive dye Rhod-4. Immunohistochemical staining revealed newly formed nerve fibers extending along the prints derived from the brain slices. While collagen-only microcontact prints stimulated nerve fiber growth, those loaded with polyornithine significantly enhanced nerve fiber outgrowth. Beta-amyloid(42) significantly increased the neurofilament-positive nerve fibers, while tau had only a weak effect. MiniRuby crystals, retrogradely transported along these newly formed nerve fibers, reached the hippocampus, while FluoVolt and Rhod-4 monitored electrical activity in newly formed nerve fibers. Our data provide evidence that intact nerve fibers can form along collagen-based microcontact prints from mouse brain slices. The Alzheimer's peptide beta-amyloid(42) stimulates this growth, hinting at a neuroprotective function when physiologically active. This "brain-on-chip" model may offer a platform for screening bioactive factors or testing drug effects on nerve fiber growth.

Emerging Evidence for the Use of Antidiabetic Drugs, Glucagon-like Peptide 1 Receptor Agonists, for the Treatment of Alzheimer's Disease.

From an epidemiological and pathophysiological point of view, Alzheimer's disease (AD) and type 2 diabetes (T2DM) should be considered 'sister' diseases. T2DM significantly increases the risk of developing AD, and the mechanisms of neuronal degeneration themselves worsen peripheral glucose metabolism in multiple ways. The pathophysiological links between the two diseases, particularly cerebral insulin resistance, which causes neuronal degeneration, are so close that AD is sometimes referred to as 'type 3 diabetes'. Although the latest news on the therapeutic front for AD is encouraging, no treatment has been shown to halt disease progression permanently. At best, the treatments slow down the progression; at worst, they are inactive, or cause worrying side effects, preventing their use on a larger scale. Therefore, it appears logical that optimizing the metabolic milieu through preventive or curative measures can also slow down the cerebral degeneration that characterizes AD. Among the different classes of hypoglycaemic drugs, glucagon-like peptide 1 receptor agonists, which are widely used in the treatment of T2DM, were shown to slow down, or even prevent, neuronal degeneration. Data from animal, preclinical, clinical phase II, cohort and large cardiovascular outcomes studies are encouraging. Of course, randomized clinical phase III studies, which are on-going, will be essential to verify this hypothesis. Thus, for once, there is hope for slowing down the neurodegenerative processes associated with diabetes, and that hope is the focus of this review.

Harnessing the Therapeutic Potential of the Nrf2/Bach1 Signaling Pathway in Parkinson's Disease.

Parkinson’s disease (PD) is the second most common neurodegenerative movement disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although a complex interplay of multiple environmental and genetic factors has been implicated, the etiology of neuronal death in PD remains unresolved. Various mechanisms of neuronal degeneration in PD have been proposed, including oxidative stress, mitochondrial dysfunction, neuroinflammation, α-synuclein proteostasis, disruption of calcium homeostasis, and other cell death pathways. While many drugs individually targeting these pathways have shown promise in preclinical PD models, this promise has not yet translated into neuroprotective therapies in human PD. This has consequently spurred efforts to identify alternative targets with multipronged therapeutic approaches. A promising therapeutic target that could modulate multiple etiological pathways involves drug-induced activation of a coordinated genetic program regulated by the transcription factor, nuclear factor E2-related factor 2 (Nrf2). Nrf2 regulates the transcription of over 250 genes, creating a multifaceted network that integrates cellular activities by expressing cytoprotective genes, promoting the resolution of inflammation, restoring redox and protein homeostasis, stimulating energy metabolism, and facilitating repair. However, FDA-approved electrophilic Nrf2 activators cause irreversible alkylation of cysteine residues in various cellular proteins resulting in side effects. We propose that the transcriptional repressor of BTB and CNC homology 1 (Bach1), which antagonizes Nrf2, could serve as a promising complementary target for the activation of both Nrf2-dependent and Nrf2-independent neuroprotective pathways. This review presents the current knowledge on the Nrf2/Bach1 signaling pathway, its role in various cellular processes, and the benefits of simultaneously inhibiting Bach1 and stabilizing Nrf2 using non-electrophilic small molecules as a novel therapeutic approach for PD.

Changes in activity of µ- and m-calpains and signs of neuroinflammation in the hippocampus and striatum of rats after single intraperitoneal injection of subseptic dose of endotoxin.

Some mechanisms of neuronal degeneration in endotoxinemia are already well described, but need to be detailed. In this study, we tested the effect of a single intraperitoneal injection of a LPS sub-septic dose (1mg/kg of animal weight) on calpain activity in the striatum and hippocampus. We showed, that in the hippocampus the day after LPS administration an increase in production of IL-1β and TNF-α mRNA, followed by elevated mRNA expression and activity of µ- and m-calpains without signs of microglia activation is observed. In striatal cells, the day after LPS injection an increase in expression of IL-1β, TNF-α, IBA-1, m-calpain and calpastatin mRNA is revealed, which only intensifies over time. The elicited changes are accompanied by a decrease in motor behavior, which can be considered as a sign of sickness behavior. In the hippocampus, 180days after LPS administration expression of TNF-α, content and activity of µ-calpain are increased. In the striatum, elevation in expression of TNF-α, IBA-1, µ- and m-calpain mRNA, with hyperactivation of only m-calpain, is observed. Significantly reduced motor activity can be a consequence of LPS-induced neuronal death. A long-lasting endotoxin activates microglia that damage neurons via proinflammation cytokines and calpain hyperactivation. The endotoxin hypothesis of neurodegeneration is unproven, but if correct, then neurodegeneration may be reduced by decreasing endotoxin-induced neuroinflammation and m-calpain hyperactivation. Therefore, the drugs, that decrease endotoxin-induced neuroinflammation and differently inhibit µ- or m-calpain, can be used to prevent or reduce the severity of neurodegeneration.

Neuronal degeneration and associated alterations in cytokine and protein in an experimental branch retinal venous occlusion model

The mechanisms of neuronal degeneration and associated acute alterations in intraretinal cytokine and protein levels remain poorly understood in variable ischaemic retinopathies such as in branch retinal vein occlusion (BRVO). Herein we investigate neuronal damage and alterations in retinal cytokines and proteins in a pig model of acute BRVO. Twelve pigs had a BRVO induced photothrombotically in both eyes. Three pigs (6 eyes) each at 2, 6, 10 and 20 days were sacrificed together with an additional 3 control (6 eyes), enucleated, retinas dissected and processed. Apoptosis in the inner retina was determined by terminal deoxyynuclotidyl transferase mediated dUTP nick end labelling (TUNEL) and histology. Expression of glial acidic fibrillary protein (GFAP), aquaporin-4 (AQP4), inward rectifier potassium channel 10 protein (Kir 4.1) encoded by KCNJ10 gene, vascular endothelial growth factor (VEGF), stromal derived growth factor-1α (SDF-1) encoded by CXCL12 gene and interleukin (IL) −6 and 8 were analysed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunohistochemistry. TUNEL labelling showed positive nuclei in the ganglion cell layer (GCL) and the inner nuclear layer (INL) which was significantly higher at 2 days after BRVO compared to other time points (p < 0.05). Analysis by RT-qPCR revealed that compared with controls, BRVO significantly increased mRNA expression of GFAP at 6, 10 and 20, AQP4 at 20, VEGF at 6, SDF-1 at 20 and IL-8 at 2 and 10 days respectively (p < 0.05): Kir 4.1 at 6, VEGF at 2 and 10, and IL-6 at 2 days were significantly decreased (p < 0.05). This study indicates that neural cell death occurs early in this acute model and the responses include inflammation and breakdown of osmohomeostasis as evidenced by the upregulation of GFAP and IL-8 and down regulation of Kir 4.1 associated with glyotic changes. Early short term VEGF upregulation seen may be related to involvement of Muller glial cells. These findings support the development of acute therapeutic strategies aimed at preservation of retinal neural cells as part of an overall management plan for BRVO.

Convection Enhanced Delivery: A Comparison of infusion characteristics in ex vivo and in vivo non-human primate brain tissue.

Convection enhanced delivery (CED) is emerging as a promising infusion toolto facilitate delivery of therapeutic agents into the brain via mechanically controlled pumps. Infusion protocols and catheter design have an important impact on delivery. CED is a valid alternative for systemic administration of agents in clinical trials for cell and gene therapies. Where gel and ex vivo models are not sufficient in modeling the disease, in vivo models allow researchers to better understand the underlying mechanisms of neuron degeneration, which is helpful in finding novel approaches to control the process or reverse the progression. Determining the risks, benefits, and efficacy of new gene therapies introduced via CED will pave a way to enter human clinical trial. The objective of this study is to compare volume distribution (Vd)/ volume infused (Vi) ratios and backflow measurements following CED infusions in ex vivo versus in vivo non-human primate brain tissue, based on infusion protocols developed in vitro. In ex vivo infusions, the first brain received 2 infusions using a balloon catheter at rates of 1 μL/min and 2 μL/min for 30 minutes. The second and third brains received infusions using a valve-tip (VT) catheter at 1 μL/min for 30 minutes. The fourth brain received a total of 45 μL infused at a rate of 1 μL/min for 15 minutes followed by 2 μL/min for 15 minutes. Imaging was performed (SPGR FA34) every 3 minutes. In the in vivo group, 4 subjects received a total of 8 infusions of 50 μL. Subjects 1 and 2 received infusions at 1.0 μL/min using a VT catheter in the left hemisphere and a smart-flow (SF) catheter in the right hemisphere. Subjects 3 and 4 each received 1 infusion in the left and right hemisphere at 1.0 μL/min. MRI calculations of Vd/Vi did not significantly differ from those obtained on post-mortem pathology. The mean measured Vd/Vi of in vivo (5.23 + /-1.67) compared to ex vivo (2.17 + /-1.39) demonstrated a significantly larger Vd/Vi for in vivo by 2.4 times (p = 0.0017). We detected higher ratios in the in vivo subjects than in ex vivo. This difference could be explained by the extra cellular space volume fraction. Studies evaluating backflow and morphology use in vivo tissue as a medium are recommended. Further investigation is warranted to evaluate the role blood pressure and heart rate may play in human CED clinical trials.

Unravelling the enigma of selective vulnerability in neurodegeneration: motor neurons resistant to degeneration in ALS show distinct gene expression characteristics and decreased susceptibility to excitotoxicity

A consistent clinical feature of amyotrophic lateral sclerosis (ALS) is the sparing of eye movements and the function of external sphincters, with corresponding preservation of motor neurons in the brainstem oculomotor nuclei, and of Onuf’s nucleus in the sacral spinal cord. Studying the differences in properties of neurons that are vulnerable and resistant to the disease process in ALS may provide insights into the mechanisms of neuronal degeneration, and identify targets for therapeutic manipulation. We used microarray analysis to determine the differences in gene expression between oculomotor and spinal motor neurons, isolated by laser capture microdissection from the midbrain and spinal cord of neurologically normal human controls. We compared these to transcriptional profiles of oculomotor nuclei and spinal cord from rat and mouse, obtained from the GEO omnibus database. We show that oculomotor neurons have a distinct transcriptional profile, with significant differential expression of 1,757 named genes (q < 0.001). Differentially expressed genes are enriched for the functional categories of synaptic transmission, ubiquitin-dependent proteolysis, mitochondrial function, transcriptional regulation, immune system functions, and the extracellular matrix. Marked differences are seen, across the three species, in genes with a function in synaptic transmission, including several glutamate and GABA receptor subunits. Using patch clamp recording in acute spinal and brainstem slices, we show that resistant oculomotor neurons show a reduced AMPA-mediated inward calcium current, and a higher GABA-mediated chloride current, than vulnerable spinal motor neurons. The findings suggest that reduced susceptibility to excitotoxicity, mediated in part through enhanced GABAergic transmission, is an important determinant of the relative resistance of oculomotor neurons to degeneration in ALS.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-1058-5) contains supplementary material, which is available to authorized users.

Futures pistes thérapeutiques médicamenteuses pour la maladie de Parkinson

Parkinson's disease is characterized by a triad of cardinal motor symptoms (bradykinesia, rigidity and tremor) resulting from the loss of dopaminergic neurons in the substantia nigra. This synucleinopathy is classified in the larger group of Lewy body disorders. Currently, these symptoms are relatively well alleviated by drugs that restore dopaminergic neurotransmission, andlor by deep brain stimulation. It is not yet possible to halt the underlying degeneration, or to treat symptoms due to non-dopaminergic neuron damage. This review examines the mechanisms of neuronal degeneration in Parkinson's disease, new targets for neuroprotection, and the mechanisms causing symptoms resistant to current treatments.

156 Motor neurones subtypes resistant to degeneration in amyotrophic lateral sclerosis show distinct synaptic characteristics across species

A consistent clinical feature of amyotrophic lateral sclerosis (ALS) is the sparing of eye movements and the function of external sphincters, with corresponding preservation of motor neurones (MNs) in the...

α-Synuclein Expression Selectively Affects Tumorigenesis in Mice Modeling Parkinson's Disease

Alpha Synuclein (α-Syn) is a protein implicated in mechanisms of neuronal degeneration in Parkinson's disease (PD). α-Syn is primarily a neuronal protein, however, its expression is found in various tumors including ovarian, colorectal and melanoma tumors. It has been hypothesized that neurodegeneration may share common mechanisms with oncogenesis. We tested whether α-Syn expression affects tumorigenesis of three types of tumors. Specifically, B16 melanoma, E0771 mammary gland adenocarcinoma and D122 Lewis lung carcinoma. For this aim, we utilized transgenic mice expression the human A53T α-Syn form. We found that the in vivo growth of B16 and E0771 but not D122 was enhanced in the A53T α-Syn mice. The effect on tumorigenesis was not detected in age-matched APP/PS1 mice, modeling Alzheimer's disease (AD), suggesting a specific effect for α-Syn- dependent neurodegeneration. Importantly, transgenic α-Syn expression was detected within the three tumor types. We further show uptake of exogenously added, purified α-Syn, by the cultured tumor cells. In accord, with the affected tumorigenesis in the young A53T α-Syn mice, over- expression of α-Syn in cultured B16 and E0771 cells enhanced proliferation, however, had no effect on the proliferation of D122 cells. Based on these results, we suggest that certain forms of α-Syn may selectively accelerate cellular mechanisms leading to cancer.

Nigrostriatal neuronal death following chronic dichlorvos exposure: crosstalk between mitochondrial impairments, α synuclein aggregation, oxidative damage and behavioral changes.

BackgroundIn recent years, several lines of evidence have shown an increase in Parkinson's disease prevalence in rural environments where pesticides are heavily used. Although, the underlying mechanism for neuronal degeneration in sporadic PD remains unknown, mitochondrial dysfunction, oxidative stress and proteasomal dysfunction are proposed as contributing factors. In this study rats were chronically and continuously exposed to the pesticide, dichlorvos to identify the molecular mechanism of nigrostaital neuronal degeneration.ResultChronic dichlorvos exposure (2.50 mg/kg b.wt.s.c/daily for 12 weeks) caused nigrostriatal dopaminergic degeneration. The degenerative changes were accompanied by a loss of 60-80% of the nigral dopamine neurons and 60-70% reduction in striatal dopamine and tyrosine hydroxylase levels. Dichlorvos exposed animals also showed α -synuclein and ubiquitin positive inclusions along with swollen, dystrophic neurites and mitochondrial abnormalities like decreased complex I&IV activities, increased mitochondrial size, axonal degeneration and presence of electron dense perinuclear cytoplasmic inclusions in the substantia nigra of rats. These animals also showed evidence of oxidative stress, including increased mitochondrial ROS levels, decreased MnSOD activity and increased lipid peroxidation. Measurable impairments in neurobehavioral indices were also observed. Notable exacerbations in motor impairments, open field and catalepsy were also evident in dichlorvos exposed animals.ConclusionAll these findings taken together indicate that chronic dichlorvos exposure may cause nigrostaital neurodegenaration and significant behavioral impairments.

Treatment strategies for Parkinson’s disease

Parkinson's disease (PD) is caused by progressive degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc), resulting in the deficiency of DA in the striatum. Thus, symptoms are developed, such as akinesia, rigidity and tremor. The aetiology of neuronal death in PD still remains unclear. Several possible mechanisms of the degeneration of dopaminergic neurons are still elusive. Various mechanisms of neuronal degeneration in PD have been proposed, including formation of free radicals, oxidative stress, mitochondrial dysfunction, excitotoxicity, calcium cytotoxicity, trophic factor deficiency, inflammatory processes, genetic factors, environmental factors, toxic action of nitric oxide, and apoptosis. All these factors interact with each other, inducing a vicious cycle of toxicity causing neuronal dysfunction, atrophy and finally cell death. Considerable evidence suggests that free radicals and oxidative stress may play key roles in the pathogenesis of PD. However, currently, drug therapy cannot completely cure the disease. DA replacement therapy with levodopa (L-Dopa), although still being a gold standard for symptomatic treatment of PD, only alleviates the clinical symptoms. Furthermore, patients usually experience severe side effects several years after the L-Dopa treatment. Until now, no therapy is available to stop or at least slow down the neurodegeneration in patients. Therefore, efforts are made not only to improve the effect of L-Dopa treatment for PD, but also to investigate new drugs with both antiparkinsonian and neuroprotective effects. Here, the advantages and limitations of current and future therapies for PD were dicussed. Current therapies include dopaminergic therapy, DA agonists, MAO-B inhibitor, COMT inhibitors, anticholinergic drugs, surgical procedures such as pallidotomy and more specifically deep brain stimulation of the globus pallidus pars interna (GPi) or subthalamic nucleus (STN), and stem cell transplantation.

Distinct patterns of gene and protein expression elicited by organophosphorus pesticides in Caenorhabditis elegans.

BackgroundThe wide use of organophosphorus (OP) pesticides makes them an important public health concern. Persistent effects of exposure and the mechanism of neuronal degeneration are continuing issues in OP toxicology. To elucidate early steps in the mechanisms of OP toxicity, we studied alterations in global gene and protein expression in Caenorhabditis elegans exposed to OPs using microarrays and mass spectrometry. We tested two structurally distinct OPs (dichlorvos and fenamiphos) and employed a mechanistically different third neurotoxicant, mefloquine, as an out-group for analysis. Treatment levels used concentrations of chemical sufficient to prevent the development of 10%, 50% or 90% of mid-vulval L4 larvae into early gravid adults (EGA) at 24 h after exposure in a defined, bacteria-free medium.ResultsAfter 8 h of exposure, the expression of 87 genes responded specifically to OP treatment. The abundance of 34 proteins also changed in OP-exposed worms. Many of the genes and proteins affected by the OPs are expressed in neuronal and muscle tissues and are involved in lipid metabolism, cell adhesion, apoptosis/cell death, and detoxification. Twenty-two genes were differentially affected by the two OPs; a large proportion of these genes encode cytochrome P450s, UDP-glucuronosyl/UDP-glucosyltransferases, or P-glycoproteins. The abundance of transcripts and the proteins they encode were well correlated.ConclusionExposure to OPs elicits a pattern of changes in gene expression in exposed worms distinct from that of the unrelated neurotoxicant, mefloquine. The functional roles and the tissue location of the genes and proteins whose expression is modulated in response to exposure is consistent with the known effects of OPs, including damage to muscle due to persistent hypercontraction, neuronal cell death, and phase I and phase II detoxification. Further, the two different OPs evoked distinguishable changes in gene expression; about half the differences are in genes involved in detoxification, likely reflecting differences in the chemical structure of the two OPs. Changes in the expression of a number of sequences of unknown function were also discovered, and these molecules could provide insight into novel mechanisms of OP toxicity or adaptation in future studies.

Identification and kainic acid-induced up-regulation of low-affinity p75 neurotrophin receptor (p75NTR) in the nigral dopamine neurons of adult rats

Parkinson's disease is a common and severe debilitating neurological disease that results from massive and progressive degenerative death of dopamine neurons in the substantia nigra, but the mechanisms of neuronal degeneration and disease progression remains largely obscure. We are interested in possible implications of low-affinity p75 neurotrophin receptor (p75NTR), which may mediate neuronal apoptosis in the central nervous system, in triggering cell death of the nigral dopamine neurons. The RT-PCR and immunohistochemistry were carried out to detect if p75NTR is expressed in these nigral neurons and up-regulated by kainic acid (KA) insult in adult rats. It revealed p75NTR-positive immunoreactivity in the substantia nigra, and co-localization of p75NTR and tyrosine hydroxylase (TH) was found in a large number of substantia nigra neurons beside confirmation of p75NTR in the choline acetyltransferase (ChAT)-positive forebrain neurons. Cell count data further indicated that about 47–100% of TH-positive nigral neurons and 98–100% of ChAT-positive forebrain neurons express p75NTR. More interestingly, significant increasing in both p75NTR mRNA and p75NTR-positive neurons occurred rapidly following KA insult in the substantia nigra of animal model. The present study has provided first evidence on p75NTR expression and KA-inducing p75NTR up-regulation in substantia nigra neurons in rodent animals. Taken together with previous data on p75NTR functions in neuronal apoptosis, this study also suggests that p75NTR may play important roles in neuronal cell survival or excitotoxic degeneration of dopamine neurons in the substantia nigra in pathogenesis of Parkinson's disease in human beings.

Mutant dynactin in motor neuron disease.

Impaired axonal transport in motor neurons has been proposed as a mechanism for neuronal degeneration in motor neuron disease. Here we show linkage of a lower motor neuron disease to a region of 4 Mb at chromosome 2p13. Mutation analysis of a gene in this interval that encodes the largest subunit of the axonal transport protein dynactin showed a single base-pair change resulting in an amino-acid substitution that is predicted to distort the folding of dynactin's microtubule-binding domain. Binding assays show decreased binding of the mutant protein to microtubules. Our results show that dysfunction of dynactin-mediated transport can lead to human motor neuron disease.

Inverse association of Pin1 and tau accumulation in Alzheimer's disease hippocampus.

Neurofibrillary degeneration, one of the pathological hallmarks of Alzheimer's disease, is not ubiquitous to all brain regions or neurons. While a high degree of vulnerability has been documented for entorhinal cortex, hippocampal and neocortical pyramidal neurons other brain structures are largely spared. Even within highly vulnerable regions such as hippocampus neurons are affected to a variable extent. The molecular basis for this selective susceptibility remains unknown. Neurofibrillary degeneration involves hyperphosphorylation of tau which critically impairs its binding capacity to microtubule and, therefore, is believed to disrupt the axonal cytoskeleton. Recently, Lu et al. [Nature (1999) 399:784] described the ability of the peptidyl-prolyl cis-trans isomerase Pin1 to recover microtubule-binding affinity and microtubule stabilisation of phosphorylated tau. In the present study, we analysed the potential involvement of Pin1 in selective vulnerability of hippocampal neurons to neurofibrillary degeneration in Alzheimer's disease. Pin1 immunoreactivity appeared as cytoplasmic granules affecting hippocampal subfields to a different extent (CA2>subiculum>CA1>CA3/CA4). Since the main markers of granulovacuolar degeneration do not co-label Pin1-immunoreactive granules, we propose that these granules may represent a new lesion in Alzheimer's disease. Neurons containing Pin1 granules were devoid of neurofibrillary tangles. Granular accumulation of Pin1 may correspond to an absence of neurofibrillary lesions in these cells and might be associated with other mechanisms of neuronal degeneration.

UVB irradiation-induced apoptosis increased in lymphocytes of Huntington's disease patients.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG repeat expansion in the IT-15 gene coding for huntingtin. The mechanism of neuronal degeneration induced by the mutant huntingtin is not known. Apoptosis may play a role in it. Huntingtin is widely expressed in the cells, so abnormalities can be expected also in non-neural tissue. We examined the susceptibility of lymphocytes from HD patients, asymptomatic carriers and normal individuals to UVB irradiation-induced apoptosis. Lymphocytes from eight HD patients and two asymptomatic carriers showed increased apoptotic cell death compared to controls. Our results suggests that sensitivity of HD cells to induced apoptosis is not restricted to neurons.

Motor neurons rapidly accumulate DNA single-strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy.

The mechanisms of neuronal degeneration in motor neuron disease are not fully understood. We tested the hypothesis that oxidative stress in vitro and axotomy in vivo induce single-strand breaks (SSB) in DNA, a form of early DNA damage, in adult motor neurons early during their degeneration. We developed and characterized a novel cell suspension system enriched in motor neurons from adult rat spinal cord ventral horn. This cell system is approximately 84% neurons, with approximately 86% of these neurons being motor neurons; approximately 72% of these motor neurons are alpha-motor neurons. Motor neuron viability in suspension is approximately 100% immediately after isolation and approximately 61% after 12 hours of incubation. During incubation, isolated motor neurons generate high levels of superoxide. We used single-cell gel electrophoresis (comet assay) to detect DNA-SSB in motor neurons. Exposure of motor neurons to nitric oxide (NO) donors (sodium nitroprusside or NONOate), H2O2, or NO donor plus H2O2 rapidly induces DNA-SSB and causes motor neuron degeneration, the occurrence of which is dose and time related, as represented by comet formation and cell loss. Motor neuron toxicity is potentiated by cotreatment with NO donor and H2O2 (at nontoxic concentrations alone). Peroxynitrite causes DNA-SSB in motor neurons. The DNA damage profiles (shown by the comet morphology and moment) of NO donors, NO donor plus H2O2, and peroxynitrite are similar. In an in vivo model of motor neuron apoptosis, DNA-SSB accumulate slowly in avulsed motor neurons before apoptotic nuclear features emerge, and the comet fingerprint is similar to NO toxicity. We conclude that motor neurons challenged by oxidative stress and axotomy accumulate DNA-SSB early in their degeneration and that the formation of peroxynitrite is involved in the mechanisms.

Mechanisms of L-cysteine neurotoxicity.

We review here the possible mechanisms of neuronal degeneration caused by L-cysteine, an odd excitotoxin. L-Cysteine lacks the omega carboxyl group required for excitotoxic actions via excitatory amino acid receptors, yet it evokes N-methyl-D-aspartate (NMDA) -like excitotoxic neuronal death and potentiates the Ca2+ influx evoked by NMDA. Both actions are prevented by NMDA antagonists. One target for cysteine effects is thus the NMDA receptor. The following mechanisms are discussed now: (1) possible increase in extracellular glutamate via release or inhibition of uptake/degradation, (2) generation of cysteine alpha-carbamate, a toxic analog of NMDA, (3) generation of toxic oxidized cysteine derivatives, (4) chelation of Zn2+ which blocks the NMDA receptor-ionophore, (5) direct interaction with the NMDA receptor redox site(s), (6) generation of free radicals, and (7) formation of S-nitrosocysteine. In addition to these, we describe another new alternative for cytotoxicity: (8) generation of the neurotoxic catecholamine derivative, 5-S-cysteinyl-3,4-dihydroxyphenylacetate (cysdopac).

Selective subcellular redistributions of protein kinase C isoforms by chemical hypoxia

The mechanisms of neuronal degeneration following hypoxia/ischemia remain undefined, but the processes include increases in neurotransmitter release, elevation of cytosolic-free calcium concentration, and changes in signal transduction pathways. Activation of the multigene family of protein kinase C (PKC) has been associated with the release of neurotransmitter and the survival of neurons. Therefore, to understand which PKC isozymes are involved in hypoxia/ischemia-induced neuronal degeneration, we examined PKC isozymes after chemical hypoxia (i.e., KCN exposure) in PC12 cells. Cell toxicity, as measured by lactate dehydrogenase (LDH) release, was increased significantly by KCN in glucose-free DMEM and was exaggerated by acute 12-O-tetradecanoyl phorbol-13-acetate (TPA) pretreatment. Under parallel conditions, KCN elevated cytosolic-free calcium ([Ca2+]i) in glucose-free but not in glucose containing DMEM, and TPA pretreatment did not exaggerate KCN's effect on [Ca2+]i. Thus, increases in [Ca2+]i are not sufficient for the synergistic toxic effect of KCN and TPA. In the glucose-free DMEM, selective PKC isozyme inhibitor Go 6976 at 10 nM completely inhibited KCN-induced LDH release and at higher concentrations (1 μM) inhibited the basal levels of LDH release. The protein levels of PKCs in the nuclear, membrane, and cytosolic fractions were measured by Western blot analysis using antibodies against specific isoforms. Two Ca2+-dependent (−α, −γ) and four Ca2+-independent (−δ, −ϵ, −ζ, and −λ) isozymes were identified and two isozymes (−β and −θ) were not detected in the subcellular fractions of PC12 cells. Treatment of the cells with TPA significantly activated translocation of conventional PKC-γ from the cytosol to the membrane and nuclear fractions and other PKC isozymes (−α, −δ, and −ϵ) from the cytosol to the membrane, but not atypical PKC-ζ and -λ. Although only the levels in the nuclear PKC-γ but not other PKC isozymes were increased significantly following KCN, the levels of cPKC-α and -γ in the membrane mainly—and those and PKC-ϵ in the nucleus—were increased when KCN was combined with TPA. In addition, this condition (TPA + KCN) did not affect the TPA insensitive atypical isozymes, PKC-ζ or -λ. Taking the results together, differential activation/translocation of PKC isozymes by KCN and TPA is important in the regulation of chemical hypoxia-induced cell injury in PC12 cells. J. Neurosci. Res. 56:668–678, 1999. © 1999 Wiley-Liss, Inc.