Human Neurodegenerative Disorders Research Articles

Quinolinic acid induced neurodegeneration in the striatum: a combined in vivo and in vitro analysis of receptor changes and microglia activation

Huntington's disease (HD) is a progressive neurodegenerative disorder, which is characterised by prominent neuronal cell loss in the basal ganglia with motor and cognitive disturbances. One of the most well-studied pharmacological models of HD is produced by local injection in the rat brain striatum of the excitotoxin quinolinic acid (QA), which produces many of the distinctive features of this human neurodegenerative disorder. Here, we report a detailed analysis, obtained both in vivo and in vitro of this pharmacological model of HD. By combining emission tomography (PET) with autoradiographic and immunocytochemical confocal laser techniques, we quantified in the QA-injected striatum the temporal behavior (from 1 to 60 days from the excitotoxic insult) of neuronal cell density and receptor availability (adenosine A(2A) and dopamine D(2) receptors) together with the degree of microglia activation. Both approaches showed a loss of adenosine A(2A) and dopamine D(2) receptors paralleled by an increase of microglial activation. This combined longitudinal analysis of the disease progression, which suggested an impairment of neurotransmission, neuronal integrity and a reversible activation of brain inflammatory processes, might represent a more quantitative approach to compare the differential effects of treatments in slowing down or reversing HD in rodent models with potential applications to human patients.

Celastrol inhibits production of nitric oxide and proinflammatory cytokines through MAPK signal transduction and NF-κB in LPS-stimulated BV-2 microglial cells

Excessive production of nitric oxide (NO) and proinflammatory cytokines from activated microglia play an important role in human neurodegenerative disorders. Here, we investigated whether celastrol, which has been used as a potent anti-inflammatory and anti-oxidative agent in Chinese medicine, attenuates excessive production of NO and proinflammatory cytokines such as TNF-alpha and IL-1betal in LPS-stimulated BV-2 cells, a mouse microglial cell line. We report here that the LPS-elicited excessive production of NO, TNF-alpha, and IL-1beta in BV-2 cells was largely inhibited in the presence of celastrol, and the attenuation of inducible iNOS and these cytokines resulted from the reduced expression of mRNAs of iNOS and these cytokines, respectively. The molecular mechanisms that underlie celastrol-mediated attenuation were the inhibition of LPS-induced phosphorylation of MAPK/ERK1/2 and the DNA binding activity of NF-kappaB in BV-2 cells. The results indicate that celastrol effectively attenuated NO and proinflammatory cytokine production via the inhibition of ERK1/2 phosphorylation and NF-kappaB activation in LPS-activated microglia. Thus, celastrol may be an effective therapeutic candidate for use in the treatment of neurodegenerative human brain disorders.

MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.

Intracellular Protein Degradation From a Vague Idea through the Lysosome and the Ubiquitin‐Proteasome System and on to Human Diseases and Drug Targeting

Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code is transcribed to RNA and translated to proteins, but how proteins are degraded has remained a neglected research area. With the discovery of the lysosome by Christian de Duve, it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis is largely nonlysosomal, but the mechanisms involved had remained obscure. The discovery of the ubiquitin-proteasome system resolved this enigma. We now recognize that ubiquitin- and proteasome-mediated degradation of intracellular proteins is involved in the regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of many human diseases, malignancies, and neurodegenerative disorders among them, which led subsequently to an increasing effort to develop mechanism-based drugs; one is already in use.

Sulfogalactosylceramides in motor and psycho-cognitive adult metachromatic leukodystrophy: relations between clinical, biochemical analysis and molecular aspects

Metachromatic leukodystrophy (MLD) is a human autosomal recessive lysosomal neurodegenerative disorder that results from the accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). During adolescence and/or adulthood, there are 2 clinical presentations. It may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis. As several lines of evidence indicate that the psychotic form of MLD could be a model of psychosis, we decided to do a pluridisciplinary study on 11 psycho-cognitive cases involving mental and psychiatric testing, in comparison with 5 adult motor cases, a biochemical study with enzyme assays and quantitative mass spectrometry of urinary sulfatides, so as to determine whether there were biochemical particularities related to the psychotic forms. For quantitative mass spectrometry (MS), a non physiological sulfatide with C17:0 fatty acid was synthesized. The major sulfatide isoforms were present in the 2 clinical forms with the following fatty acids and sphingoid bases: C22:1/d18:1, and /or C22:0/d18:2 ( m/ z 862.5), C22:0 (OH)/d18:1 ( m/ z 878,5), C24:0/d18:1 and / or C24:0/C23:1(OH)/d18:2 ( m/ z 890,3), C24:0 (OH)/d18:1( m/ z 906.5). We had shown previously that there were different ASA mutations in the psychiatric adult form (heterozygous I179S) versus the adult motor form (homozygous P426L). We show here that there were no relations with the level of ASA and with the mass spectrometric study of the sulfatide isoforms which were identical in the 2 clinical forms.

P.1.c.043 Evidence for the involvement of monoaminergic system in the antidepressant-like effect of magnesium

AMPA receptors are fast ligand-gated members of glutamate receptors in neuronal and many types of non-neuronal cells. The heterotetramer complexes are assembled from four subunits (GluR1–4) in region-, development- and function-selective patterns. Each subunit contains three extracellular domains (a large amino terminal domain, an agonist-binding domain and a transducer domain), and three transmembrane segments with a loop (pore forming domain), as well as the intracellular carboxy terminal tail (traffic and conductance regulatory domain). The binding of the agonist (excitatory amino acids and their derivatives) initiates conformational realignments, which transmit to the transducer domain and membrane spanning segments to gate the channel permeable to Na+, K+ and more or less to Ca2+. Several 2,3-benzodiazepines act as non-competitive antagonists of the AMPA receptor (termed also negative allosteric modulators), which are thought to bind to the transducer domains and inhibit channel gating. Analysing their effects in vitro, it has been possible to recognize a structure–activity relationship, and to describe the critical parts of the molecules involved in their action at AMPA receptors. Blockade of AMPA receptors can protect the brain from apoptotic and necrotic cell death by preventing neuronal excitotoxicity during pathophysiological activation of glutamatergic neurons. Animal experiments provided evidence for the potential usefulness of non-competitive AMPA antagonists in the treatment of human ischemic and neurodegenerative disorders including stroke, multiple sclerosis, Parkinson's disease, periventricular leukomalacia and motoneuron disease. 2,3-Benzodiazepine AMPA antagonists can protect against seizures, decrease levodopa-induced dyskinesia in animal models of Parkinson's disease demonstrating their utility for the treatment of a variety of CNS disorders.

Tau phosphorylation sites work in concert to promote neurotoxicity in vivo.

Tau is a microtubule binding protein implicated in a number of human neurodegenerative disorders, including Alzheimer's disease. Phosphorylation of serine-proline/threonine-proline sites, targeted by proline-directed kinases, coincides temporally with neurodegeneration in the human diseases. Recently, we demonstrated that this unique group of serines and threonines has a critical role in controlling tau toxicity in a Drosophila model of tauopathy. Here, we use a combination of genetic and biochemical approaches to examine these sites individually and to determine which of them is primarily responsible for controlling tau neurotoxicity. Despite the importance placed on individual phosphoepitopes and their contributions to disease pathogenesis, our results indicate that no single phosphorylation residue plays a dominant role in controlling tau toxicity. These findings suggest that serine-proline/threonine-proline sites cooperate to mediate neurodegeneration in vivo.

Targeting ubiquitin specific proteases for drug discovery

Deregulation of the ubiquitin-proteasome system has been implicated in the pathogenesis of many human diseases, including cancer, neurodegenerative disorders and viral diseases. The recent approval of the proteasome inhibitor bortezomib (Velcade ®) for the treatment of multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. A promising alternative to targeting the proteasome itself would be to interact at the level of the upstream, ubiquitin conjugation/deconjugation system to generate more specific, less toxic anticancer agents. Ubiquitin specific proteases (USP) are de-ubiquitinating enzymes which remove ubiquitin from specific protein substrates and allow protein salvage from proteasome degradation, regulation of protein localization or activation. Due to their protease activity and their involvement in several pathologies, USPs are emerging as potential target sites for pharmacological interference in the ubiquitin regulatory machinery. We will review here this class of enzymes from target validation to small molecule drug discovery.

Tauopathy models and human neuropathology: similarities and differences

Much of our current understanding of the pathogenic mechanisms in human neurodegenerative disorders has been derived from animal studies. As such, transgenic mouse models have significantly contributed to the development of novel pathogenic concepts underlying human tauopathies, a group of diseases comprising various forms of neurodegenerative disorders including Alzheimer's disease, corticobasal degeneration, argyrophilic grain disease, progressive supranuclear palsy, and Pick's disease as well as hereditary fronto-temporal dementia with parkinsonism linked to chromosome 17. Here, we will review in vivo models of human tauopathies with particular preference to transgenic mouse models. Strengths and limitations of these models in recapitulating the complex pathogenesis of tauopathies will be discussed.

Visceral Neuropathy and Intestinal Pseudo-Obstruction in a Murine Model of a Nuclear Inclusion Disease

Intestinal dysmotility is a component of many neurodegenerative disorders, including some characterized by neuronal intranuclear inclusions. PrP-SCA7-92Q transgenic mice phenocopy many aspects of the human polyglutamine neurodegenerative disorder spinocerebellar ataxia type 7 (SCA7). The enteric neuropathology of PrP-SCA7-92Q mice was investigated after observing that they develop signs of intestinal pseudo-obstruction. Gastrointestinal transit of radio-opaque pellets through presymptomatic and symptomatic PrP-SCA7-92Q mice and nontransgenic littermates was compared. Gross, microscopic, and ultrastructural studies were conducted, along with histologic and whole mount immunohistochemistry, to identify intranuclear inclusions and quantify subsets of enteric neurons. Immunoblot analysis was performed to confirm selective loss of particular neuronal populations. A subset of cholinergic enteric ganglion cells in PrP-SCA7-92Q mice harbor nuclear inclusions composed of transgene-derived ataxin-7, which contains a pathogenic polyglutamine expansion. These animals die between 15 and 20 weeks of age with intestinal distension and enterocolitis. Signs of disease are preceded by selective loss of nitric oxide synthase-positive neurons (which lack nuclear inclusions), loss of nerve fibers in the myenteric nerve plexus, and delayed gastrointestinal transit. Cholinergic neurons, including those with inclusions, are spared. PrP-SCA7-92Q mice may be useful models for human intestinal pseudoobstruction, particularly visceral neuropathies with neuronal intranuclear inclusions. Loss of inclusion-free inhibitory neurons supports the hypothesis that inclusions may be neuroprotective or coincidental, as opposed to harbingers of neuron death. Because enteric neuropathology in PrP-SCA7-92Q animals is easily missed by routine histopathology, quantitative immunohistochemical approaches may be required to recognize analogous forms of human enteric neuropathy.

Doppel Induces Degeneration of Cerebellar Purkinje Cells Independently of Bax

Doppel (Dpl) is a prion protein paralog that causes neurodegeneration when expressed ectopically in the brain. To investigate the cellular mechanism underlying this effect, we analyzed Dpl-expressing transgenic mice in which the gene for the proapoptotic protein Bax had been deleted. We found that Bax deletion does not alter either clinical symptoms or Purkinje cell degeneration in Dpl transgenic mice. In addition, we observed that degenerating Purkinje cells in these animals do not display DNA fragmentation or caspase-3 activation. Our results suggest that non-Bax-dependent pathways mediate the toxic effects of Dpl in Purkinje cells, highlighting a possible role for nonapoptotic mechanisms in the death of these neurons.

2,3-Benzodiazepine-type AMPA receptor antagonists and their neuroprotective effects

AMPA receptors are fast ligand-gated members of glutamate receptors in neuronal and many types of non-neuronal cells. The heterotetramer complexes are assembled from four subunits (GluR1–4) in region-, development- and function-selective patterns. Each subunit contains three extracellular domains (a large amino terminal domain, an agonist-binding domain and a transducer domain), and three transmembrane segments with a loop (pore forming domain), as well as the intracellular carboxy terminal tail (traffic and conductance regulatory domain). The binding of the agonist (excitatory amino acids and their derivatives) initiates conformational realignments, which transmit to the transducer domain and membrane spanning segments to gate the channel permeable to Na+, K+ and more or less to Ca2+. Several 2,3-benzodiazepines act as non-competitive antagonists of the AMPA receptor (termed also negative allosteric modulators), which are thought to bind to the transducer domains and inhibit channel gating. Analysing their effects in vitro, it has been possible to recognize a structure–activity relationship, and to describe the critical parts of the molecules involved in their action at AMPA receptors. Blockade of AMPA receptors can protect the brain from apoptotic and necrotic cell death by preventing neuronal excitotoxicity during pathophysiological activation of glutamatergic neurons. Animal experiments provided evidence for the potential usefulness of non-competitive AMPA antagonists in the treatment of human ischemic and neurodegenerative disorders including stroke, multiple sclerosis, Parkinson's disease, periventricular leukomalacia and motoneuron disease. 2,3-Benzodiazepine AMPA antagonists can protect against seizures, decrease levodopa-induced dyskinesia in animal models of Parkinson's disease demonstrating their utility for the treatment of a variety of CNS disorders.

SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis.

A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.

Mutations in Cytochrome c Oxidase Subunit VIa Cause Neurodegeneration and Motor Dysfunction in Drosophila

Mitochondrial dysfunction is involved in many neurodegenerative disorders in humans. Here we report mutations in a gene (designated levy) that codes for subunit VIa of cytochrome c oxidase (COX). The mutations were identified by the phenotype of temperature-induced paralysis and showed the additional phenotypes of decreased COX activity, age-dependent bang-induced paralysis, progressive neurodegeneration, and reduced life span. Germ-line transformation using the levy(+) gene rescued the mutant flies from all phenotypes including neurodegeneration. The data from levy mutants reveal a COX-mediated pathway in Drosophila, disruption of which leads to mitochondrial encephalomyopathic effects including neurodegeneration, motor dysfunction, and premature death. The data present the first case of a mutation in a nuclear-encoded structural subunit of COX that causes mitochondrial encephalomyopathy rather than lethality, whereas several previous attempts to identify such mutations have not been successful. The levy mutants provide a genetic model to understand the mechanisms underlying COX-mediated mitochondrial encephalomyopathies and to explore possible therapeutic interventions.

Aggregation of cellular prion protein is initiated by proximity‐induced dimerization

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.

Effect of proteasomal inhibition by MG-132 on inclusion body formation in astrocytes

The small heat shock proteins (sHSPs), HSP25/27 and alpha B-crystallin, are more prominent in glia than in neurons, act as molecular chaperones and specifically interact with cytoskeletal elements. They represent prominent constituents in inclusion bodies originating in astrocytes and oligodendrocytes, and have been described to be involved in Alexander's disease, a human neurodegenerative disorder with astrocytic inclusions called Rosenthal fibres (RF). These protein aggregates also contain GFAP (glial fibrillary acidic protein) and ubiquitin, indicating that inclusion body formation might be causally related to an impairment of the ubiquitin proteasome pathway (UPS).

Influence of autophagy genes on ion-channel-dependent neuronal degeneration in Caenorhabditis elegans

Necrotic cell death is a common feature in numerous human neurodegenerative disorders. In the nematode Caenorhabditis elegans, gain-of-function mutations in genes that encode specific ion channel subunits such as the degenerins DEG-1 and MEC-4, and the acetylcholine receptor subunit DEG-3 lead to necrotic-like degeneration of a subset of neurons. Neuronal demise caused by ion channel hyperactivity is accompanied by intense degradation of cytoplasmic contents, dramatic membrane infolding and vacuole formation; however, the cellular pathways underlying such processes remain largely unknown. Here we show that the function of three autophagy genes, whose yeast and mammalian orthologs are implicated in cytoplasmic self-degradation, membrane trafficking and the cellular response to starvation, contributes to ion-channel-dependent neurotoxicity in C. elegans. Inactivation of unc-51, bec-1 and lgg-1, the worm counterparts of the yeast autophagy genes Atg1, Atg6 and Atg8 respectively, partially suppresses degeneration of neurons with toxic ion channel variants. We also demonstrate that the TOR-kinase-mediated signaling pathway, a nutrient sensing system that downregulates the autophagy gene cascade, protects neurons from undergoing necrotic cell death, whereas nutrient deprivation promotes necrosis. Our findings reveal a role for autophagy genes in neuronal cell loss in C. elegans.

Retrolinkin, a membrane protein, plays an important role in retrograde axonal transport

Retrograde axonal transport plays an important role in the maintenance of neuronal functions, but the mechanism is poorly defined partly because the constituents of the retrograde transport system and their interactions have yet to be elucidated. Of special interest is how dynein/dynactin motor proteins interact with membrane cargoes. Here, we report that an endosomal vesicle protein, termed retrolinkin, functions as a receptor tethering vesicles to dynein/dynactin through BPAG1n4. Retrolinkin, a membrane protein highly enriched in neuronal endosomes, binds directly to BPAG1n4. Deletion of retrolinkin membrane-association domains disrupts retrograde vesicular transport, recapitulating the BPAG1 null phenotype. We propose that retrolinkin acts with BPAG1n4 to specifically regulate retrograde axonal transport. Our work lays the foundation for understanding fundamental issues of axonal transport and provides insights into the molecular mechanisms underlying human neurodegenerative disorders.

Sept4, a Component of Presynaptic Scaffold and Lewy Bodies, Is Required for the Suppression of α-Synuclein Neurotoxicity

In Parkinson disease (PD), alpha-synuclein aggregates called Lewy bodies often involve and sequester Septin4 (Sept4), a polymerizing scaffold protein. However, the pathophysiological significance of this phenomenon is unclear. Here, we show the physiological association of Sept4 with alpha-synuclein, the dopamine transporter, and other presynaptic proteins in dopaminergic neurons; mice lacking Sept4 exhibit diminished dopaminergic neurotransmission due to scarcity of these presynaptic proteins. These data demonstrate an important role for septin scaffolds in the brain. In transgenic mice that express human alpha-synuclein(A53T) (a mutant protein responsible for familial PD), loss of Sept4 significantly enhances neuropathology and locomotor deterioration. In this PD model, insoluble deposits of Ser129-phosphorylated alpha-synuclein(A53T) are negatively correlated with the dosage of Sept4. In vitro, direct association with Sept4 protects alpha-synuclein against self-aggregation and Ser129 phosphorylation. Taken together, these data show that Sept4 may be involved in PD as a dual susceptibility factor, as its insufficiency can diminish dopaminergic neurotransmission and enhance alpha-synuclein neurotoxicity.

Mutant prion protein D202N associated with familial prion disease is retained in the endoplasmic reticulum and forms 'curly' intracellular aggregates.

Transmissible Spongiform Encephalopathies are fatal neurodegenerative disorders of humans and animals that are familial, sporadic, and infectious in nature. Familial disorders of humans include Gerstmann-Straussler-Scheinker disease (GSS), familial Creutzfeldt-Jakob disease (CJD), and fatal familial insomnia, and result from point mutations in the prion protein gene. Although neurotoxicity in familial cases is believed to result from a spontaneous change in conformation of mutant prion protein (PrP) to the pathogenic PrP-scrapie (PrPSc) form, emerging evidence indicates otherwise. We have investigated the processing and metabolism of mutant PrP D202N (PrP202N) in cell models to elucidate possible mechanisms of cytotoxicity. In this report, we demonstrate that PrP202N expressed in human neuroblastoma cells fails to achieve a mature conformation following synthesis and accumulates in the endoplasmic reticulum as 'curly' aggregates. In addition, PrP202N cells show increased sensitivity to free radicals, indicating that neuronal susceptibility to oxidative damage may account for the neurotoxicity observed in cases of GSS resulting from PrP D202N mutation.