Studying neurodegenerative diseases in culture models

Abstract

Neurodegenerative diseases are pathological conditions that have an insidious onset and chronic progression. Different models have been established to study these diseases in order to understand their underlying mechanisms and to investigate new therapeutic strategies. Although various in vivo models are currently in use, in vitro models might provide important insights about the pathogenesis of these disorders and represent an interesting approach for the screening of potential pharmacological agents. In the present review, we discuss various in vitro and ex vivo models of neurodegenerative disorders in mammalian cells and tissues.

Neurodegenerative diseases; in vitro models; ex vivo models; neurons; neuroglia

Background

Neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, affect millions of people around the world. Unfortunately, the pathogenesis of these chronic neurodegenerative diseases is not fully understood, and current treatments do not stop or slow down progression of these pathological conditions. Therefore, different in vivo, ex vivo, and in vitro models have been generated. In vitro models of these pathological conditions offer advantages over in vivo models in several aspects. First, it is possible to study the role of isolated cells of one particular type in an environment that simulates the disease and to investigate mechanisms of a possible deleterious or protective role of specific molecules and compounds. Second, screening for potential actions of drugs is also facilitated. In this sense, in vitro models of neurodegenerative processes have been used to provide important clues about mechanisms of the diseases and potential pharmacological targets. In the present review, we discuss in vitro and ex vivo models of chronic neurodegenerative diseases using cells or tissues.

Parkinson's disease

PD is a slowly progressive neurodegenerative disease clinically characterized by motor impairment, namely bradykinesia, rigidity, resting tremor, and postural instability.¹ Synaptic and axonal degeneration within the striatum followed by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) leads to reduced levels of dopamine in the nigrostriatal circuitry.² Besides dopaminergic cell loss, intracellular formation of Lewy bodies and Lewy neurites, consisting predominantly of aggregated alpha-synuclein (αSyn), has been suggested to be crucial in the pathogenesis of this disease.³ Moreover, genetic factors contribute to the pathogenesis of PD.⁴ To date, more than 16 loci and 11 associated genes have been identified. Among these, mutations in the gene for αSyn were the first ones to be mapped.⁵ On the cellular level, research in PD focuses on protein aggregation, neurotoxicity, increased oxidative stress, excitotoxicity, mitochondrial dysfunction, and defects in the protein degradation machinery (including the ubiquitin-proteasomal system and autophagy pathways).⁶

Several cell culture systems have been employed to study these possible disease processes. But what would be the perfect cell? A homogeneous cell culture system that is easy to handle would be preferable. Cells should be easy to expand in order to generate large numbers of neuronal precursor cells. Next, these cells should be able to be transferred from a proliferative into a post-mitotic state. Finally, these cells should be easily directed towards a post-mitotic state in a synchronized manner with a mature neuronal (dopaminergic) phenotype. One cell culture model that will surely play an important role in PD research and that already combines many of the aforementioned aspects consists of dopaminergic neurons derived from human induced pluripotent stem cells redirected from human fibroblasts.⁷ However, we will not include human induced pluripotent stem cells in this review, because their usage is still hampered by very labor-intensive and costly procedures. Some problems, such as the low absolute yield of differentiated dopaminergic neurons and low homogeneity, are of high research interest and we would like to refer the interested reader to the very comprehensive review by Studer.⁸

Primary midbrain dopaminergic neurons are suitable to study dopaminergic cell survival and neurite retraction as well as regeneration. Usually, embryonic midbrain neurons from embryonic day 14 (E14) are dissected.⁹ A high yield of dopaminergic neurons can be obtained, which can be exposed to various neurodegenerative stimuli. Several neurotoxins are employed to study neurodegeneration. In particular, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP⁺) are widely accepted to induce neurotoxicity. Both neurotoxins are thought to induce dopaminergic toxicity by intra- and extracellular oxidation, hydrogen peroxide formation, and direct inhibition of the mitochondrial respiratory chain.¹⁰ On the one hand, this cell model is very suitable to study methods of neurodegeneration and neurite retraction; on the other hand, possible neurorestorative capacities by pharmacological compounds and the underlying mechanisms can be nicely dissected. For example, inhibition of rho kinase mediated by fasudil promotes the survival of rat primary midbrain neurons after addition of MPP⁺ to the culture dish via the Akt survival pathway.¹¹ As a read-out for cell survival, cell number can be counted in this cell culture system. Other possibilities include, for example, the MTT assay, determination of adenylate kinase in the supernatant, or fluorescence-activated cell sorter (FACS) analysis for annexin V and propidium iodide.

Since axonal loss seems to be an early event in PD pathogenesis, analysis of the neurite network can also be performed and may be suitable as a good read-out for neurite preservation. An interesting approach to study neurite regeneration has been described by Tönges et al. Neurite processes were mechanically transected with a thin silicone scraper, treated with MPP+, and finally the length of neurites was determined using ImageJ software.¹¹,¹² However, primary midbrain neurons are not easy to prepare; they are time-consuming and hard to transfect. Therefore, different cell lines were generated.

A commonly human cell line used in PD research is the HEK293 (human embryonic kidney 293) cell line. These cells can be easily transfected (e.g., via calcium phosphate, liposome based, electroporation). In one paper, the kinetics of αSyn aggregation was studied with respect to aggregation formation. Increased expression of wild-type αSyn was shown to result in the formation of cytoplasmic aggregates.¹³ Time-lapse imaging illustrated how cells form and accumulate aggregates of αSyn in HEK293 cells.¹⁴ HEK293 cell line is also a suitable model system to study the effect of αSyn mutations and other PD associated genes. The expression of a mutant A53T form of αSyn caused an increased susceptibility to dopamine.¹³ Recently, it could be demonstrated that overexpression of leucine-rich repeat kinase 2 (LRRK2) does not result in altered gene expression in HEK293 cells.¹⁵ Mutations in LRRK2 are strongly associated with late-onset autosomal dominant PD, and HEK293 may be suitable to go for candidate pharmacological screening for LRRK2 inhibitors.¹⁶ Moreover, mechanisms of possible in vitro transfer of αSyn and its modified species may be studied in this cell line.¹⁷

Despite the common usage of HEK293 cells in PD research, there are some drawbacks, including the fact that these cells lack a neuronal phenotype. Another cell line that is widely used in the field of PD research is the SH-SY5Y cell line, which is derived from human neuroblastoma cells. These cells are widely used to study mechanisms of neurodegeneration. For example, overexpression of wild-type human αSyn was shown to promote inclusion formation in SH-SY5Y cells.¹⁸ Moreover, extracellular addition of αSyn oligomers caused transmembrane seeding of αSyn aggregation in a dose- and time-dependent manner.¹⁹ However, SH-SY5Y cells are hard to differentiate into a post-mitotic mature dopaminergic state.²⁰

Several other human cell lines mainly derived from embryonic teratocarcinomas (NT2, hNT) are currently used, and they can be directed towards a post-mitotic neuronal phenotype.²¹,²² The human H4 neuroglioma cell line has been used to study the oligomerization of intracellular αSyn by fluorescence lifetime imaging (FLIM) for the first time.²³ Moreover, the role of αSyn in the autophagy pathway has been addressed in this cell line.²⁴ It could be shown that dysfunction of the autophagy pathway may lead to exosome-mediated release of αSyn oligomers in order to clear these toxin αSyn species.²⁵ However, all these cell lines are derived from tumorous cells and only moderately show a distinct neuronal phenotype. Thus, we would finally like to address here the Lund human mesencephalic (LUHMES) cells. LUHMES cells were derived from 8-week-old human fetal ventral mesencephalic cells. To induce immortalization and thereby continuous proliferation, these cells were transformed based on the LINXv-myc vector with tetracycline-regulated v-myc expression.²⁶ This vector also contains a tetracycline transactivator that enhances the expression of v-myc from a minimal promoter from human cytomegalovirus (CMV) fused to the tetracycline operator sequence. Addition of tetracycline inactivates the transactivator and thereby abolishes v-myc expression. Supplementation with GDNF and cAMP induces a dopaminergic phenotype after 5 days of differentiation.²⁷ Differentiated LUHMES cells showed a high degree of dopaminergic phenotype, including release of dopamine and neuronal electric properties.²⁸,²⁹ The LUHMES cell line has been widely used to study dopamine-related cell death mechanisms.²⁷,²⁹,³⁰ A drawback of this cell line is that classical transfection methods showed very low transfection efficiency. Thus, a lentiviral approach to efficiently transfect these cells is necessary.

Recent reports support the hypothesis that extracellular αSyn plays an important role in PD-associated neurodegenerative processes.³¹,³² These findings suggest that extracellular αSyn released by neurons may also modulate microglial and astrocytic activity. Both glial types may respond to extracellular αSyn by increased expression of inflammatory mediators. In particular, inflammation in PD has been recognized recently not only as a mere bystander in the disease process but also as an important disease modifying or even accelerating factor. There is accumulating evidence for inflammatory processes in the progression of PD derived from 1) serum and cerebrospinal fluid (CSF) analyses, 2) genetic analyses, and 3) epidemiological studies.³³ In post-mortem studies of PD patients, expression of pro-inflammatory cytokines was elevated in the striatum of PD patients, and activated microglia was observed within the SNpc, respectively.³³,³⁴ Astrocytes and microglia cultures will be addressed in a separate topic below.

Alzheimer's disease

AD is a slowly progressive neurodegenerative disorder and the most common cause of dementia in the elderly. The neuropsychological profile of AD includes deficits in episodic memory, language, semantic knowledge, visuospatial abilities, executive functions (i.e., panning, organization, etc.), and apraxia.³⁵ The brain regions involved early in the course of the disease are the entorhinal cortex and the CA1 region of the hippocampus, followed by limbic structures and, at later stages, all isocortical areas.³⁶ The neurodegenerative process is characterized by early damage to synapses with retrograde degeneration of axons and eventual atrophy of the dendritic tree. In fact, loss of synapses is the best correlate of the cognitive impairment in patients with AD.³⁷,³⁸ Neuropathological changes include abundant extracellular amyloid plaques and neurofibrillary tangles, comprised of hyperphosphorylated tau.³⁹ Deciphering mechanisms leading to neuronal dysfunction and cell loss are the main advantages of in vitro model systems. Different neuronal cell lines are commonly used for neuronal in vitro culture system, such as PC12, HEK293, and SH-SY5Y cell lines. These cells can be transfected with wild-type amyloid-precursor proteins, tau, or mutant forms of these molecules. In addition to cell lines, primary cortical and hippocampal cultures play a valuable tool in AD research. The addition of amyloid-beta to the medium of primary neuronal cells induces apoptosis.⁴⁰,⁴¹

It is widely accepted that glial cells also contribute to the pathogenesis of AD. It has been shown that, besides neuronal loss, reactive astrocytes and activated microglial cells can be associated with amyloid plaques and neurofibrillary tangles.⁴²-⁴⁴ Although amyloid-beta itself can be toxic to neurons, it also activates microglia, leading to neuronal damage.⁴⁵ Below, we discuss the role of glial cells in AD and PD and make a brief discussion of how these cells might be used for the investigation of these two pathological conditions.

Microglia and astrocytes in AD and PD

Microglia, the phagocytic innate immune cells of the central nervous system (CNS), continuously survey the local microenvironment. Activated microglia can be morphologically distinguished from “resting” microglia, because activated microglia have larger cell bodies as well as thicker and shorter processes.⁴⁶ Detection of pathogens or adverse patterns is accomplished by a vast array of highly conserved pattern-recognition receptors, including Toll-like receptors (TLRs). Stimulation of TLRs results in the activation of well-characterized signaling pathways, e.g., nuclear factor κB, and eventually leads to subsequent transcriptional activation of pro-inflammatory genes and to the production of reactive oxygen species.⁴⁵

Primary microglial cells from rat or mice are commonly used to study inflammatory processes. For instance, primary microglial cells can be isolated from cerebral cortices of 1-day-old Wistar rats.⁴⁷-⁵¹ It is important to take extreme care to avoid lipopolysaccharide contamination, thus to keep microglia in a resting or “surveying” state instead of an activated state. Floating microglia can be harvested from 10- to 14-day-old mixed astroglial and microglial primary cultures. Finally, the purity of the microglial culture should be determined. Several microglial markers can be obtained to perform immunocytochemistry or FACS analysis, e.g., Iba1, CD68 (ED1), CD11b (OX-42), tomato lectin, or isolectin-B4. Primary microglial cultures have been used to study whether and by what means extracellular αSyn can activate microglial cells. Indeed, consistent and permanent microglial activation and subsequent production of pro-inflammatory cytokines have been shown in primary microglial cells.⁵²-⁵⁸ In particular, TLR4 may be crucial in activating microglial cells and may be involved in phagocytosis.⁵⁴ Another recently published study showed that oligomeric αSyn may interact and activate TLR2 in microglial cells.⁵⁹

The production of inflammatory mediators might contribute to the formation of amyloid-beta plaques.⁶⁰ Also, microglia of PS1-APP transgenic mice, a mouse model for AD, express increased amounts of cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF) α in comparison to their WT littermates, probably contributing to increased neurodegeneration.⁶¹ Moreover, primary microglial cells may be used to study inflammatory processes and anti-inflammatory approaches. For example, the role of prostaglandins and underlying cell signaling after activation of lipopolysaccharide led to novel insights.⁶²-⁶⁶

Since AD and PD are age-related disorders and microglia may change their functional properties in the aging brain,⁶⁷ protocols are in demand for the isolation of microglia from adult rodents. A few protocols exist; however, presently, literature is scarce on this topic.⁶⁸-⁷⁰

Besides the primary microglia cell system, one microglial cell line is widely used, i.e., the BV-2 cell line. Microglial cells from C57Bl/6 were immortalized with v-myc.⁷¹ The BV-2 cell was recently characterized, and transcriptome and proteome analysis revealed a high similarity to primary microglial cells.⁷² Since BV-2 are easy to culture, they are a valuable tool to study not only inflammatory processes,⁷² but also phagocytosis.⁷³ In addition, astrocytes may contribute to the activation of microglial cells and vice versa.⁷⁴

Reactive astrogliosis, characterized by hypertrophy of astrocytic processes and soma as well as increased proliferation accompanied by progressive changes in gene expression, is generally moderate in human post-mortem tissue of PD patients. Astrocytes are the most abundant cells in the CNS, and show a wide variety of functions including regulation of blood flow and synaptic function, but may also play an important role in mediating neuroinflammation in neurodegenerative diseases. Indeed, astrocytes play an important role in initiating and regulating CNS immune response through the release of pro-inflammatory cytokines and chemokines.⁷⁵ Recently, it could be shown that αSyn is directly transferred from neurons to primary astrocytes in vitro. Interestingly, αSyn was uptaken by astrocytes via endocytosis and showed an increase in TNFα gene expression.⁷⁶

Primary astrocytes cultures are relatively easy to prepare. Astrocytes can be obtained from every region of the CNS and at any age, although the optimal time point would be in rodents from 2-3 days postnatal when astrogenesis is at its peak.⁷⁷ Several astrocytes isolation protocol exist.⁷⁸-⁸⁰ However, a caution needs to be taken when dealing with astrocytic cultures because these cells may be “contaminated” with a high amount of microglia, oligodendrocytes, neurons, and endothelial cells.⁸¹ Thus, it is important to use specific markers for the cell types. Commonly employed astrocyte markers are GFAP, GLAST, vimentin, glutamine synthetase, glutamate transporter 1, aldehyde dehydrogenase 1 L1, and S100beta.⁸²-⁸⁴ To determine the percentage of microglial cells, immunocytochemical or FACS analysis for common microglial markers should be performed. Several methods can be used to reduce the number of microglial cells. First, frequent medium changes, shaking, and subculturing all reduce the number of microglial cells. Secondly, laminin enhances astroglial growth and inhibits microglial growth.⁸⁵,⁸⁶ Also, application of cytosine arabinoside (Ara-C) or L-leucine methyl ester may effectively deprive the astrocytes cultures from microglial cells. In addition to primary cell cultures, a few astroglial cell lines exist, such as the human U373 astrocytoma cell line.⁸⁷-⁸⁹

Huntington's disease

HD is an autosomal dominant inherited neurodegenerative disease characterized by progressive motor abnormalities, psychiatric symptoms, and cognitive decline. The cause of the disease is accepted as a CAG repeat expansion in the huntingtin gene, resulting in a long stretch of polyglutamine (PolyQ) in the encoded protein, huntingtin (Htt).⁹⁰ This mutant huntingtin (mHtt) contains more than 40 glutamine repeats. Thirty-six to 40 glutamine repeats are associated with an increased risk for developing HD and a slower progression of the pathology.⁹¹ HD is characterized predominantly by degeneration of striatal and cortical neurons, although other regions can also be affected.⁹¹

Few in vitro models have been developed to study important hallmarks of HD, allowing the investigation of key intracellular mechanism involved in the disease, as well as the identification of novel pharmacological targets. Considering the role of mHtt in the pathogenesis of HD, this protein has been used as a main tool for the study of HD in vitro. Increased frequency of aggregates is associated with toxicity in in vitro models of HD.⁹²,⁹³ It has been shown that expression of the truncated mHtt in HD models resembles the disease process at a delayed stage of PolyQ toxicity. Conversely, expression of the full-length mHtt would be more representative of the entire process observed in the disease.⁹⁴

Many aspects of the pathological features observed in HD, such as the role of mHtt protein, can be studied in neuronal cells. Examples of these cells are the rat pheochromocytoma (PC-12), the mouse Neuro2a (N2a), and the human SH-SY5Y.⁹⁵ PC-12 cells can be transfected with different PolyQ-expanded huntingtin constructs.⁹⁶,⁹⁷ For example, transfection of these cells with HD exon-1 protein with expanded polyglutamine (150Q) reveals mHtt localization in the nucleus, as well as altered morphology, multiple gene expression, and decreased viability.⁹⁸ Moreover, these cells can also be transfected with a construct (pCDNA3-1-GFP-HtEx1-104Q) that expresses HtEx1 with 104 glutamines fused with GFP under the control of a cytomegalovirus-based promoter.⁹⁹,¹⁰⁰ Various other transfections of PC12 have been performed, e.g., with the exon 1 region of the Htt gene with 109 CAG repeats¹⁰¹ and with an ecdysone-inducible protein comprising the first 17 amino acids of huntingtin plus 103 glutamines fused with enhanced GFP (htt103Q-EGFP).¹⁰²,¹⁰³ N2a neuroblastoma cells can also be transfected with different types of mHtt. For example, N2a stably expressing truncated htt with expanded 150Q tracts lead mainly to cytoplasmic aggregates formation.¹⁰⁴

The ST14A cells are derived from E14 rat striatum primordial cells that exhibit characteristics of medium-size spiny neurons and can also be transfected with mHtt.¹⁰⁵-¹⁰⁸ Another important cell model is the mouse-rat neuroblastoma-glioma hybrid cell line NG108-15 that exhibits neuronal properties after differentiation, allowing the expression of mHtt over many days.¹⁰⁹-¹¹¹ Besides that, the immortalized rat hippocampal neuronal cell line (HN33) is another type of cell used because the hippocampus is one of the brain regions affected in HD.¹¹²-¹¹⁴ Expression of PolyQ-expanded huntingtin in these cells has been shown to induce apoptosis.¹¹²

Although many cell lines have been used, they might reveal different aspects in comparison with primary cells. Therefore, primary neurons prepared from HD transgenic mice are frequently used: neocortical or striatal cultures from HdhQ111 mice that have 111 CAG repeats in exon 1 of the mHtt gene¹¹⁵-¹¹⁹; neostriatal cultures of the YAC46 (668 line) and YAC72 (2511 line) mice, which express the full-length mutant huntingtin containing 46 or 72 glutamine repeats (46Q or 72Q)¹²⁰; YAC128 (line 55) mice expressing full-length human mHtt containing 128 CAG repeats¹²¹; transgenic BACHD mice that express a full-length mHtt with 97 glutamine repeats.¹²²

Besides the neuronal cells, mHtt may also be transfected to non-neuronal cells,⁹⁵ such as HeLa cells,¹²³-¹²⁶ human embryonic kidney cell-line 293T (HEK293T),⁹³,¹²³,¹²⁶,¹²⁷ and monkey kidney cell lines (COS-7).¹⁰⁷,¹²⁸,¹²⁹

An interesting approach has been obtained with acute transfection of rat corticostriatal brain slices with DNA constructs derived from the human mHtt.¹³⁰ This model has an advantage in comparison with the isolated cells since it maintains the resident interaction between the cells, which is important for the pathogenesis of HD.¹³⁰ Importantly, this model might be used for the screening of potential compounds for the treatment of HD.

Non-genetic animal models of HD, which use chemical substances, have also been used. The 3-nitropropionic acid and quinolinic acid (QA) are used as excitotoxic agents in animal models of HD. The first compound is a mitochondrial toxin that induces neurotoxicity by irreversible inhibition of succinate dehydrogenase, a key enzyme located at the internal mitochondrial membrane and responsible for succinate oxidation to fumarate. Conversely, QA is an agonist of the N-methyl d-aspartate type glutamate receptors.¹³¹ The excitotoxicity induced by these agents are studied in organotypic striatal, corticostriatal or sagittal hypothalamic slice cultures,¹³¹-¹³³ as well as hippocampal slices from the transgenic mice R6/2.¹³⁴,¹³⁵

Amyotrophic lateral sclerosis

ALS, also known as Charcot's or Lou Gehrig's disease, is characterized by a degeneration of cortical motor neurons and anterior horn cells of the spinal cord. This leads to muscle atrophy, loss of muscle control, and death resulting from respiratory failure, generally within 3-5 years of diagnosis.

Different studies have shown that oxidative stress plays a major role in the pathogenesis of this disease, classified as a rare familial form, which frequently exhibits mutations of the superoxide dismutase 1 (SOD1) gene.¹³⁶,¹³⁷

Considering that the disease affects motor neurons, different cell lines with the characteristics of these neurons can be used to study ALS. Moreover, these cells can be transfected with mutant SOD-1. Examples of cell lines include mouse neural hybrid cell line (MN-1), which expresses motor neuron features and high affinity glutamate transporters,¹³⁸ and mouse motor neuron hybridoma line NSC-34, a hybridoma cell line derived from the fusion of neuroblastoma cells with mice spinal cord cells.¹³⁹-¹⁴³ Moreover, primary cells, like the mouse primary spinal cord culture,¹⁴⁴,¹⁴⁵ are also used.

The pathogenesis of ALS involves not only neurons, but also other cell types, such as microglia and astrocytes.¹⁴⁶ Therefore, cell lines can be transfected with mSOD1, or primary cultures can be produced from transgenic animals. Interestingly, it has been shown that expression of mSOD1 in microglia enhances the release of inflammatory mediators, augmenting its potential to induce neurotoxicity in comparison with wtSOD1.¹³⁹,¹⁴⁷-¹⁵² It has also been shown that transfection of astrocytes with mSOD1 induced toxicity to motoneurons in a co-culture model.¹⁵³,¹⁵⁴

Similar to other in vitro models of neurodegenerative disorders, organotypic rat spinal cord slice cultures,¹⁵⁵-¹⁵⁸ as well as post-mortem samples of brain and spinal cord from ALS patients,¹⁴³,¹⁵⁵ are frequently used.

Conclusion

In the present review, we discussed the possibilities of using cells and tissues in the investigation of neurodegenerative disorders. Importantly, these models might offer advantages in various aspects discussed along the text. Moreover, they complement in vivo studies that investigate the mechanisms involved in the pathogenesis of neurodegeneration.

Soraya Wilke Saliba receives research grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Antonio Carlos Pinheiro de Oliveira receives research grants from Programa Primeiros Projetos (PPP) - Fundação de Amparo è Pesquisa do Estado de Minas Gerais (FAPEMIG) (protocol no. CBB-APQ-04389-10), from Programa de Apoio a Núcleos Emergentes de Pesquisa (PRONEM) - FAPEMIG (protocol no. CBB-APQ-04625-10), and from Pró-Reitoria de Pesquisa at Universidade Federal de Minas Gerais (UFMG).

References

• ¹
  Fahn S. Description of Parkinson's disease as a clinical syndrome. Ann N Y Acad Sci. 2003;991:1-14.
• ²
  Burke RE, O'Malley K. Axon degeneration in Parkinson's disease. Exp Neurol. 2013;246:72-83.
• ³
  Lashuel HA, Overk CR, Oueslati A, Masliah E. The many faces of alpha-synuclein: from structure and toxicity to therapeutic target. Nat Rev Neurosci. 2013;14:38-48.
• ⁴
  Corti O, Lesage S, Brice A. What genetics tells us about the causes and mechanisms of Parkinson's disease. Physiol Rev. 2011;91:1161-218.
• ⁵
  Polymeropoulos MH, Higgins JJ, Golbe LI, Johnson WG, Ide SE, Di Iorio G, et al. Mapping of a gene for Parkinson's disease to chromosome 4q21-q23. Science. 1996;274:1197-9.
• ⁶
  Schulz JB. Update on the pathogenesis of Parkinson's disease. J Neurol. 2008;255:3-7.
• ⁷
  Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663-76.
• ⁸
  Studer L. Derivation of dopaminergic neurons from pluripotent stem cells. Prog Brain Res. 2012;200:243-63.
• ⁹
  Lingor P, Unsicker K, Krieglstein K. Midbrain dopaminergic neurons are protected from radical induced damage by GDF-5 application. Short communication. J Neural Transm. 1999;106:139-44.
• ¹⁰
  Blum D, Torch S, Lambeng N, Nissou M, Benabid AL, Sadoul R, et al. Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson's disease. Prog Neurobiol. 2001;65:135-72.
• ¹¹
  Tonges L, Frank T, Tatenhorst L, Saal KA, Koch JC, Szego EM, et al. Inhibition of rho kinase enhances survival of dopaminergic neurons and attenuates axonal loss in a mouse model of Parkinson's disease. Brain. 2012;135:3355-70.
• ¹²
  Tonges L, Planchamp V, Koch JC, Herdegen T, Bahr M, Lingor P. JNK isoforms differentially regulate neurite growth and regeneration in dopaminergic neurons in vitro. J Mol Neurosci. 2011;45:284-93.
• ¹³
  Tabrizi SJ, Orth M, Wilkinson JM, Taanman JW, Warner TT, Cooper JM, et al. Expression of mutant alpha-synuclein causes increased susceptibility to dopamine toxicity. Hum Mol Genet. 2000;9:2683-9.
• ¹⁴
  Opazo F, Krenz A, Heermann S, Schulz JB, Falkenburger BH. Accumulation and clearance of alpha-synuclein aggregates demonstrated by time-lapse imaging. J Neurochem. 2008;106:529-40.
• ¹⁵
  Devine MJ, Kaganovich A, Ryten M, Mamais A, Trabzuni D, Manzoni C, et al. Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue. PLoS One. 2011;6:e22489.
• ¹⁶
  Deng X, Dzamko N, Prescott A, Davies P, Liu Q, Yang Q, et al. Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2. Nat Chem Biol. 2011;7:203-5.
• ¹⁷
  Hansen C, Angot E, Bergstrom AL, Steiner JA, Pieri L, Paul G, et al. alpha-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells. J Clin Invest. 2011;121:715-25.
• ¹⁸
  Tofaris GK, Layfield R, Spillantini MG. alpha-synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome. FEBS Lett. 2001;509:22-6.
• ¹⁹
  Danzer KM, Krebs SK, Wolff M, Birk G, Hengerer B. Seeding induced by alpha-synuclein oligomers provides evidence for spreading of alpha-synuclein pathology. J Neurochem. 2009;111:192-203.
• ²⁰
  Constantinescu R, Constantinescu AT, Reichmann H, Janetzky B. Neuronal differentiation and long-term culture of the human neuroblastoma line SH-SY5Y. J Neural Transm Suppl. 200717-28.
• ²¹
  Pleasure SJ, Page C, Lee VM. Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. J Neurosci. 1992;12:1802-15.
• ²²
  Podrygajlo G, Song Y, Schlesinger F, Krampfl K, Bicker G. Synaptic currents and transmitter responses in human NT2 neurons differentiated in aggregate culture. Neurosci Lett. 2010;468:207-10.
• ²³
  Klucken J, Outeiro TF, Nguyen P, McLean PJ, Hyman BT. Detection of novel intracellular alpha-synuclein oligomeric species by fluorescence lifetime imaging. Faseb J. 2006;20:2050-7.
• ²⁴
  Klucken J, Poehler AM, Ebrahimi-Fakhari D, Schneider J, Nuber S, Rockenstein E, et al. Alpha-synuclein aggregation involves a bafilomycin A 1-sensitive autophagy pathway. Autophagy. 2012;8:754-66.
• ²⁵
  Danzer KM, Kranich LR, Ruf WP, Cagsal-Getkin O, Winslow AR, Zhu L, et al. Exosomal cell-to-cell transmission of alpha synuclein oligomers. Mol Neurodegener. 2012;7:42.
• ²⁶
  Hoshimaru M, Ray J, Sah DW, Gage FH. Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene. Proc Natl Acad Sci U S A. 1996;93:1518-23.
• ²⁷
  Lotharius J, Falsig J, van Beek J, Payne S, Dringen R, Brundin P, et al. Progressive degeneration of human mesencephalic neuron-derived cells triggered by dopamine-dependent oxidative stress is dependent on the mixed-lineage kinase pathway. J Neurosci. 2005;25:6329-42.
• ²⁸
  Scholz D, Poltl D, Genewsky A, Weng M, Waldmann T, Schildknecht S, et al. Rapid, complete and large-scale generation of post-mitotic neurons from the human LUHMES cell line. J Neurochem. 2011;119:957-71.
• ²⁹
  Schildknecht S, Poltl D, Nagel DM, Matt F, Scholz D, Lotharius J, et al. Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells. Toxicol Appl Pharmacol. 2009;241:23-35.
• ³⁰
  Depboylu C, Hollerhage M, Schnurrbusch S, Brundin P, Oertel WH, Schrattenholz A, et al. Neuregulin-1 receptor tyrosine kinase ErbB4 is upregulated in midbrain dopaminergic neurons in Parkinson disease. Neurosci Lett. 2012;531:209-14.
• ³¹
  Li JY, Englund E, Holton JL, Soulet D, Hagell P, Lees AJ, et al. Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation. Nat Med. 2008;14:501-3.
• ³²
  Desplats P, Lee HJ, Bae EJ, Patrick C, Rockenstein E, Crews L, et al. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of alpha-synuclein. Proc Natl Acad Sci U S A. 2009;106:13010-5.
• ³³
  Hirsch EC, Hunot S. Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol. 2009;8:382-97.
• ³⁴
  McGeer PL, Itagaki S, Boyes BE, McGeer EG. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology. 1988;38:1285-91.
• ³⁵
  Weintraub S, Wicklund AH, Salmon DP. The neuropsychological profile of Alzheimer disease. Cold Spring Harb Perspect Med. 2012;2:a006171.
• ³⁶
  Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol. 1991;82:239-59.
• ³⁷
  DeKosky ST, Scheff SW. Synapse loss in frontal cortex biopsies in Alzheimer's disease: correlation with cognitive severity. Ann Neurol. 1990;27:457-64.
• ³⁸
  Scheff SW, Price DA, Schmitt FA, DeKosky ST, Mufson EJ. Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology. 2007;68:1501-8.
• ³⁹
  Serrano-Pozo A, Frosch MP, Masliah E, Hyman BT. Neuropathological alterations in Alzheimer disease. Cold Spring Harb Perspect Med. 2011;1:a006189.
• ⁴⁰
  Loo DT, Copani A, Pike CJ, Whittemore ER, Walencewicz AJ, Cotman CW. Apoptosis is induced by beta-amyloid in cultured central nervous system neurons. Proc Natl Acad Sci U S A. 1993;90:7951-5.
• ⁴¹
  Nilsen J, Chen S, Irwin RW, Iwamoto S, Brinton RD. Estrogen protects neuronal cells from amyloid beta-induced apoptosis via regulation of mitochondrial proteins and function. BMC Neurosci. 2006;7:74.
• ⁴²
  Schlachetzki JC, Hull M. Microglial activation in Alzheimer's disease. Curr Alzheimer Res. 2009;6:554-63.
• ⁴³
  Serrano-Pozo A, Mielke ML, Gomez-Isla T, Betensky RA, Growdon JH, Frosch MP, et al. Reactive glia not only associates with plaques but also parallels tangles in Alzheimer's disease. Am J Pathol. 2011;179:1373-84.
• ⁴⁴
  Vehmas AK, Kawas CH, Stewart WF, Troncoso JC. Immune reactive cells in senile plaques and cognitive decline in Alzheimer's disease. Neurobiol Aging. 2003;24:321-31.
• ⁴⁵
  Block ML, Zecca L, Hong JS. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci. 2007;8:57-69.
• ⁴⁶
  Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. Physiology of microglia. Physiol Rev. 2011;91:461-553.
• ⁴⁷
  Akundi RS, Candelario-Jalil E, Hess S, Hull M, Lieb K, Gebicke-Haerter PJ, et al. Signal transduction pathways regulating cyclooxygenase-2 in lipopolysaccharide-activated primary rat microglia. Glia. 2005;51:199-208.
• ⁴⁸
  Keller M, Jackisch R, Seregi A, Hertting G. Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures. Neurochem Int. 1985;7:655-65.
• ⁴⁹
  Seregi A, Keller M, Jackisch R, Hertting G. Comparison of the prostanoid synthesizing capacity in homogenates from primary neuronal and astroglial cell cultures. Biochem Pharmacol. 1984;33:3315-8.
• ⁵⁰
  Gebicke-Haerter PJ, Bauer J, Schobert A, Northoff H. Lipopolysaccharide-free conditions in primary astrocyte cultures allow growth and isolation of microglial cells. J Neurosci. 1989;9:183-94.
• ⁵¹
  Giulian D, Baker TJ. Characterization of ameboid microglia isolated from developing mammalian brain. J Neurosci. 1986;6:2163-78.
• ⁵²
  Chen CY, Weng YH, Chien KY, Lin KJ, Yeh TH, Cheng YP, et al. (G2019S) LRRK2 activates MKK4-JNK pathway and causes degeneration of SN dopaminergic neurons in a transgenic mouse model of PD. Cell Death Differ. 2012;19:1623-33.
• ⁵³
  Cao S, Standaert DG, Harms AS. The gamma chain subunit of Fc receptors is required for alpha-synuclein-induced pro-inflammatory signaling in microglia. J Neuroinflammation. 2012;9:259.
• ⁵⁴
  Fellner L, Irschick R, Schanda K, Reindl M, Klimaschewski L, Poewe W, et al. Toll-like receptor 4 is required for alpha-synuclein dependent activation of microglia and astroglia. Glia. 2013;61:349-60.
• ⁵⁵
  Rojanathammanee L, Murphy EJ, Combs CK. Expression of mutant alpha-synuclein modulates microglial phenotype in vitro. J Neuroinflammation. 2011;8:44.
• ⁵⁶
  Klegeris A, Pelech S, Giasson BI, Maguire J, Zhang H, McGeer EG, et al. Alpha-synuclein activates stress signaling protein kinases in THP-1 cells and microglia. Neurobiol Aging. 2008;29:739-52.
• ⁵⁷
  Zhang W, Wang T, Pei Z, Miller DS, Wu X, Block ML, et al. Aggregated alpha-synuclein activates microglia: a process leading to disease progression in Parkinson's disease. FASEB J. 2005;19:533-42.
• ⁵⁸
  Reynolds AD, Glanzer JG, Kadiu I, Ricardo-Dukelow M, Chaudhuri A, Ciborowski P, et al. Nitrated alpha-synuclein-activated microglial profiling for Parkinson's disease. J Neurochem. 2008;104:1504-25.
• ⁵⁹
  Kim C, Ho DH, Suk JE, You S, Michael S, Kang J, et al. Neuron-released oligomeric alpha-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia. Nat Commun. 2013;4:1562.
• ⁶⁰
  Blasko I, Stampfer-Kountchev M, Robatscher P, Veerhuis R, Eikelenboom P, Grubeck-Loebenstein B. How chronic inflammation can affect the brain and support the development of Alzheimer's disease in old age: the role of microglia and astrocytes. Aging Cell. 2004;3:169-76.
• ⁶¹
  Hickman SE, Allison EK, El Khoury J. Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice. J Neurosci. 2008;28:8354-60.
• ⁶²
  de Oliveira AC, Candelario-Jalil E, Langbein J, Wendeburg L, Bhatia HS, Schlachetzki JC, et al. Pharmacological inhibition of Akt and downstream pathways modulates the expression of COX-2 and mPGES-1 in activated microglia. J Neuroinflammation. 2012;9:2.
• ⁶³
  Wendeburg L, de Oliveira AC, Bhatia HS, Candelario-Jalil E, Fiebich BL. Resveratrol inhibits prostaglandin formation in IL-1beta-stimulated SK-N-SH neuronal cells. J Neuroinflammation. 2009;6:26.
• ⁶⁴
  Bhatia HS, Candelario-Jalil E, de Oliveira AC, Olajide OA, Martinez-Sanchez G, Fiebich BL. Mangiferin inhibits cyclooxygenase-2 expression and prostaglandin E2 production in activated rat microglial cells. Arch Biochem Biophys. 2008;477:253-8.
• ⁶⁵
  de Oliveira AC, Candelario-Jalil E, Bhatia HS, Lieb K, Hull M, Fiebich BL. Regulation of prostaglandin E2 synthase expression in activated primary rat microglia: evidence for uncoupled regulation of mPGES-1 and COX-2. Glia. 2008;56:844-55.
• ⁶⁶
  Hull M, Muksch B, Akundi RS, Waschbisch A, Hoozemans JJ, Veerhuis R, et al. Amyloid beta peptide (25-35) activates protein kinase C leading to cyclooxygenase-2 induction and prostaglandin E2 release in primary midbrain astrocytes. Neurochem Int. 2006;48:663-72.
• ⁶⁷
  Streit WJ, Miller KR, Lopes KO, Njie E. Microglial degeneration in the aging brain--bad news for neurons? Front Biosci. 2008;13:3423-38.
• ⁶⁸
  Cardona AE, Huang D, Sasse ME, Ransohoff RM. Isolation of murine microglial cells for RNA analysis or flow cytometry. Nat Protoc. 2006;1:1947-51.
• ⁶⁹
  Baker CA, Martin D, Manuelidis L. Microglia from Creutzfeldt-Jakob disease-infected brains are infectious and show specific mRNA activation profiles. J Virol. 2002;76:10905-13.
• ⁷⁰
  Nikodemova M, Watters JJ. Efficient isolation of live microglia with preserved phenotypes from adult mouse brain. J Neuroinflammation. 2012;9:147.
• ⁷¹
  Blasi E, Barluzzi R, Bocchini V, Mazzolla R, Bistoni F. Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. J Neuroimmunol. 1990;27:229-37.
• ⁷²
  Henn A, Lund S, Hedtjarn M, Schrattenholz A, Porzgen P, Leist M. The suitability of BV2 cells as alternative model system for primary microglia cultures or for animal experiments examining brain inflammation. ALTEX. 2009;26:83-94.
• ⁷³
  Hirt UA, Leist M. Rapid, noninflammatory and PS-dependent phagocytic clearance of necrotic cells. Cell Death Differ. 2003;10:1156-64.
• ⁷⁴
  Saijo K, Winner B, Carson CT, Collier JG, Boyer L, Rosenfeld MG, et al. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell. 2009;137:47-59.
• ⁷⁵
  Esen N, Tanga FY, DeLeo JA, Kielian T. Toll-like receptor 2 (TLR2) mediates astrocyte activation in response to the Gram-positive bacterium Staphylococcus aureus. J Neurochem. 2004;88:746-58.
• ⁷⁶
  Lee HJ, Suk JE, Patrick C, Bae EJ, Cho JH, Rho S, et al. Direct transfer of alpha-synuclein from neuron to astroglia causes inflammatory responses in synucleinopathies. J Biol Chem. 2010;285:9262-72.
• ⁷⁷
  Lim R, Bosch EP. Isolation of astrocytes and Schwann cells for culture. In: Coon PM, editor. Methods in neurosciences. San Diego: Academic Press; 1990. p. 47-55.
• ⁷⁸
  Booher J, Sensenbrenner M. Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology. 1972;2:97-105.
• ⁷⁹
  McCarthy KD, de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 1980;85:890-902.
• ⁸⁰
  Heinrich C, Gascon S, Masserdotti G, Lepier A, Sanchez R, Simon-Ebert T, et al. Generation of subtype-specific neurons from postnatal astroglia of the mouse cerebral cortex. Nat Protoc. 2011;6:214-28.
• ⁸¹
  Saura J. Microglial cells in astroglial cultures: a cautionary note. J Neuroinflammation. 2007;4:26.
• ⁸²
  Barres BA. The mystery and magic of glia: a perspective on their roles in health and disease. Neuron. 2008;60:430-40.
• ⁸³
  Kimelberg HK. Functions of mature mammalian astrocytes: a current view. Neuroscientist. 2010;16:79-106.
• ⁸⁴
  Kimelberg HK. The problem of astrocyte identity. Neurochem Int. 2004;45:191-202.
• ⁸⁵
  Milner R, Campbell IL. Cytokines regulate microglial adhesion to laminin and astrocyte extracellular matrix via protein kinase C-dependent activation of the alpha6beta1 integrin. J Neurosci. 2002;22:1562-72.
• ⁸⁶
  Saura J, Angulo E, Ejarque A, Casado V, Tusell JM, Moratalla R, et al. Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia. J Neurochem. 2005;95:919-29.
• ⁸⁷
  Waschbisch A, Fiebich BL, Akundi RS, Schmitz ML, Hoozemans JJ, Candelario-Jalil E, et al. Interleukin-1 beta-induced expression of the prostaglandin E-receptor subtype EP3 in U373 astrocytoma cells depends on protein kinase C and nuclear factor-kappaB. J Neurochem. 2006;96:680-93.
• ⁸⁸
  Lieb K, Kaltschmidt C, Kaltschmidt B, Baeuerle PA, Berger M, Bauer J, et al. Interleukin-1 beta uses common and distinct signaling pathways for induction of the interleukin-6 and tumor necrosis factor alpha genes in the human astrocytoma cell line U373. J Neurochem. 1996;66:1496-503.
• ⁸⁹
  Machein U, Lieb K, Hull M, Fiebich BL. IL-1 beta and TNF alpha, but not IL-6, induce alpha 1-antichymotrypsin expression in the human astrocytoma cell line U373 MG. Neuroreport. 1995;6:2283-6.
• ⁹⁰
  Landles C, Bates GP. Huntingtin and the molecular pathogenesis of Huntington's disease. Fourth in molecular medicine review series. EMBO Rep. 2004;5:958-63.
• ⁹¹
  Bano D, Zanetti F, Mende Y, Nicotera P. Neurodegenerative processes in Huntington's disease. Cell Death Dis. 2011;2:e228.
• ⁹²
  Hackam AS, Hodgson JG, Singaraja R, Zhang T, Gan L, Gutekunst CA, et al. Evidence for both the nucleus and cytoplasm as subcellular sites of pathogenesis in Huntington's disease in cell culture and in transgenic mice expressing mutant huntingtin. Philos Trans R Soc Lond B Biol Sci. 1999;354:1047-55.
• ⁹³
  Hackam AS, Singaraja R, Wellington CL, Metzler M, McCutcheon K, Zhang T, et al. The influence of huntingtin protein size on nuclear localization and cellular toxicity. J Cell Biol. 1998;141:1097-105.
• ⁹⁴
  Dong X, Zong S, Witting A, Lindenberg KS, Kochanek S, Huang B. Adenovirus vector-based in vitro neuronal cell model for Huntington's disease with human disease-like differential aggregation and degeneration. J Gene Med. 2012;14:468-81.
• ⁹⁵
  Cisbani G, Cicchetti F. An in vitro perspective on the molecular mechanisms underlying mutant huntingtin protein toxicity. Cell Death Dis. 2012;3:e382.
• ⁹⁶
  Scotter EL, Goodfellow CE, Graham ES, Dragunow M, Glass M. Neuroprotective potential of CB1 receptor agonists in an in vitro model of Huntington's disease. Br J Pharmacol. 2010;160:747-61.
• ⁹⁷
  Song C, Perides G, Liu YF. Expression of full-length polyglutamine-expanded huntingtin disrupts growth factor receptor signaling in rat pheochromocytoma (PC12) cells. J Biol Chem. 2002;277:6703-7.
• ⁹⁸
  Li SH, Cheng AL, Li H, Li XJ. Cellular defects and altered gene expression in PC12 cells stably expressing mutant huntingtin. J Neurosci. 1999;19:5159-72.
• ⁹⁹
  Kazantsev A, Preisinger E, Dranovsky A, Goldgaber D, Housman D. Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells. Proc Natl Acad Sci U S A. 1999;96:11404-9.
• ¹⁰⁰
  Muchowski PJ, Ning K, D'Souza-Schorey C, Fields S. Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment. Proc Natl Acad Sci U S A. 2002;99:727-32.
• ¹⁰¹
  Huang CL, Yang JM, Wang KC, Lee YC, Lin YL, Yang YC, et al. Gastrodia elata prevents huntingtin aggregations through activation of the adenosine A(2)A receptor and ubiquitin proteasome system. J Ethnopharmacol. 2011;138:162-8.
• ¹⁰²
  Apostol BL, Kazantsev A, Raffioni S, Illes K, Pallos J, Bodai L, et al. A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila. Proc Natl Acad Sci U S A. 2003;100:5950-5.
• ¹⁰³
  Wang J, Pfleger CM, Friedman L, Vittorino R, Zhao W, Qian X, et al. Potential application of grape derived polyphenols in Huntington's disease. Transl Neurosci. 2010;1:95-100.
• ¹⁰⁴
  Zemskov EA, Jana NR, Kurosawa M, Miyazaki H, Sakamoto N, Nekooki M, et al. Pro-apoptotic protein kinase C delta is associated with intranuclear inclusions in a transgenic model of Huntington's disease. J Neurochem. 2003;87:395-406.
• ¹⁰⁵
  Cattaneo E, Conti L. Generation and characterization of embryonic striatal conditionally immortalized ST14A cells. J Neurosci Res. 1998;53:223-34.
• ¹⁰⁶
  Ehrlich ME, Conti L, Toselli M, Taglietti L, Fiorillo E, Taglietti V, et al. ST14A cells have properties of a medium-size spiny neuron. Exp Neurol. 2001;167:215-26.
• ¹⁰⁷
  Ossato G, Digman MA, Aiken C, Lukacsovich T, Marsh JL, Gratton E. A two-step path to inclusion formation of huntingtin peptides revealed by number and brightness analysis. Biophys J. 2010;98:3078-85.
• ¹⁰⁸
  Rigamonti D, Bauer JH, De-Fraja C, Conti L, Sipione S, Sciorati C, et al. Wild-type huntingtin protects from apoptosis upstream of caspase-3. J Neurosci. 2000;20:3705-13.
• ¹⁰⁹
  Holzmann C, Maueler W, Petersohn D, Schmidt T, Thiel G, Epplen JT, et al. Isolation and characterization of the rat huntingtin promoter. Biochem J. 1998;336:227-34.
• ¹¹⁰
  Lunkes A, Mandel JL. A cellular model that recapitulates major pathogenic steps of Huntington's disease. Hum Mol Genet. 1998;7:1355-61.
• ¹¹¹
  Ravache M, Weber C, Merienne K, Trottier Y. Transcriptional activation of REST by Sp1 in Huntington's disease models. PLoS One. 2010;5:e14311.
• ¹¹²
  Liu YF. Expression of polyglutamine-expanded huntingtin activates the SEK1-JNK pathway and induces apoptosis in a hippocampal neuronal cell line. J Biol Chem. 1998;273:28873-7.
• ¹¹³
  Liu YF, Dorow D, Marshall J. Activation of MLK2-mediated signaling cascades by polyglutamine-expanded huntingtin. J Biol Chem. 2000;275:19035-40.
• ¹¹⁴
  Song C, Zhang Y, Parsons CG, Liu YF. Expression of polyglutamine-expanded huntingtin induces tyrosine phosphorylation of N-methyl-D-aspartate receptors. J Biol Chem. 2003;278:33364-9.
• ¹¹⁵
  Ribeiro FM, Paquet M, Ferreira LT, Cregan T, Swan P, Cregan SP, et al. Metabotropic glutamate receptor-mediated cell signaling pathways are altered in a mouse model of Huntington's disease. J Neurosci. 2010;30:316-24.
• ¹¹⁶
  Sarantos MR, Papanikolaou T, Ellerby LM, Hughes RE. Pizotifen activates ERK and provides neuroprotection in vitro and in vivo in models of Huntington's disease. J Huntingtons Dis. 2012;1:195-210.
• ¹¹⁷
  Snider BJ, Moss JL, Revilla FJ, Lee CS, Wheeler VC, Macdonald ME, et al. Neocortical neurons cultured from mice with expanded CAG repeats in the huntingtin gene: unaltered vulnerability to excitotoxins and other insults. Neuroscience. 2003;120:617-25.
• ¹¹⁸
  Trettel F, Rigamonti D, Hilditch-Maguire P, Wheeler VC, Sharp AH, Persichetti F, et al. Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. Hum Mol Genet. 2000;9:2799-809.
• ¹¹⁹
  Ventura I, Russo MT, De Nuccio C, De Luca G, Degan P, Bernardo A, et al. hMTH1 expression protects mitochondria from Huntington's disease-like impairment. Neurobiol Dis. 2012;49C:148-58.
• ¹²⁰
  Zeron MM, Fernandes HB, Krebs C, Shehadeh J, Wellington CL, Leavitt BR, et al. Potentiation of NMDA receptor-mediated excitotoxicity linked with intrinsic apoptotic pathway in YAC transgenic mouse model of Huntington's disease. Mol Cell Neurosci. 2004;25:469-79.
• ¹²¹
  Milnerwood AJ, Kaufman AM, Sepers MD, Gladding CM, Zhang L, Wang L, et al. Mitigation of augmented extrasynaptic NMDAR signaling and apoptosis in cortico-striatal co-cultures from Huntington's disease mice. Neurobiol Dis. 2012;48:40-51.
• ¹²²
  Doria JG, Silva FR, de Souza JM, Vieira LB, Carvalho TG, Reis HJ, et al. Metabotropic glutamate receptor 5 positive allosteric modulators are neuroprotective in a mouse model of Huntington's disease. Br J Pharmacol. 2013;169:909-21.
• ¹²³
  Iwata A, Riley BE, Johnston JA, Kopito RR. HDAC6 and microtubules are required for autophagic degradation of aggregated huntingtin. J Biol Chem. 2005;280:40282-92.
• ¹²⁴
  Majumder P, Chattopadhyay B, Mazumder A, Das P, Bhattacharyya NP. Induction of apoptosis in cells expressing exogenous Hippi, a molecular partner of huntingtin-interacting protein Hip1. Neurobiol Dis. 2006;22:242-56.
• ¹²⁵
  Raychaudhuri S, Sinha M, Mukhopadhyay D, Bhattacharyya NP. HYPK, a huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal huntingtin with 40 glutamines in neuro2a cells and exhibits chaperone-like activity. Hum Mol Genet. 2008;17:240-55.
• ¹²⁶
  Sancho M, Herrera AE, Gortat A, Carbajo RJ, Pineda-Lucena A, Orzaez M, et al. Minocycline inhibits cell death and decreases mutant huntingtin aggregation by targeting Apaf-1. Hum Mol Genet. 2011;20:3545-53.
• ¹²⁷
  Cooper JK, Schilling G, Peters MF, Herring WJ, Sharp AH, Kaminsky Z, et al. Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture. Hum Mol Genet. 1998;7:783-90.
• ¹²⁸
  Ho LW, Brown R, Maxwell M, Wyttenbach A, Rubinsztein DC. Wild type huntingtin reduces the cellular toxicity of mutant huntingtin in mammalian cell models of Huntington's disease. J Med Genet. 2001;38:450-2.
• ¹²⁹
  Martindale D, Hackam A, Wieczorek A, Ellerby L, Wellington C, McCutcheon K, et al. Length of huntingtin and its polyglutamine tract influences localization and frequency of intracellular aggregates. Nat Genet. 1998;18:150-4.
• ¹³⁰
  Reinhart PH, Kaltenbach LS, Essrich C, Dunn DE, Eudailey JA, DeMarco CT, et al. Identification of anti-inflammatory targets for Huntington's disease using a brain slice-based screening assay. Neurobiol Dis. 2011;43:248-56.
• ¹³¹
  Colle D, Hartwig JM, Soares FA, Farina M. Probucol modulates oxidative stress and excitotoxicity in Huntington's disease models in vitro. Brain Res Bull. 2012;87:397-405.
• ¹³²
  Pallier PN, Maywood ES, Zheng Z, Chesham JE, Inyushkin AN, Dyball R, et al. Pharmacological imposition of sleep slows cognitive decline and reverses dysregulation of circadian gene expression in a transgenic mouse model of Huntington's disease. J Neurosci. 2007;27:7869-78.
• ¹³³
  Storgaard J, Kornblit BT, Zimmer J, Gramsbergen JB. 3-Nitropropionic acid neurotoxicity in organotypic striatal and corticostriatal slice cultures is dependent on glucose and glutamate. Exp Neurol. 2000;164:227-35.
• ¹³⁴
  Hay DG, Sathasivam K, Tobaben S, Stahl B, Marber M, Mestril R, et al. Progressive decrease in chaperone protein levels in a mouse model of Huntington's disease and induction of stress proteins as a therapeutic approach. Hum Mol Genet. 2004;13:1389-405.
• ¹³⁵
  Smith DL, Portier R, Woodman B, Hockly E, Mahal A, Klunk WE, et al. Inhibition of polyglutamine aggregation in R6/2 HD brain slices-complex dose-response profiles. Neurobiol Dis. 2001;8:1017-26.
• ¹³⁶
  Hadzhieva M, Kirches E, Wilisch-Neumann A, Pachow D, Wallesch M, Schoenfeld P, et al. Dysregulation of iron protein expression in the G93A model of amyotrophic lateral sclerosis. Neuroscience. 2013;230:94-101.
• ¹³⁷
  Lima IV, Bastos LF, Limborco-Filho M, Fiebich BL, de Oliveira AC. Role of prostaglandins in neuroinflammatory and neurodegenerative diseases. Mediators Inflamm. 2012;2012:946813.
• ¹³⁸
  Boston-Howes W, Williams EO, Bogush A, Scolere M, Pasinelli P, Trotti D. Nordihydroguaiaretic acid increases glutamate uptake in vitro and in vivo: therapeutic implications for amyotrophic lateral sclerosis. Exp Neurol. 2008;213:229-37.
• ¹³⁹
  D'Ambrosi N, Finocchi P, Apolloni S, Cozzolino M, Ferri A, Padovano V, et al. The proinflammatory action of microglial P2 receptors is enhanced in SOD1 models for amyotrophic lateral sclerosis. J Immunol. 2009;183:4648-56.
• ¹⁴⁰
  Ferri A, Fiorenzo P, Nencini M, Cozzolino M, Pesaresi MG, Valle C, et al. Glutaredoxin 2 prevents aggregation of mutant SOD1 in mitochondria and abolishes its toxicity. Hum Mol Genet. 2010;19:4529-42.
• ¹⁴¹
  Liu R, Li B, Flanagan SW, Oberley LW, Gozal D, Qiu M. Increased mitochondrial antioxidative activity or decreased oxygen free radical propagation prevent mutant SOD1-mediated motor neuron cell death and increase amyotrophic lateral sclerosis-like transgenic mouse survival. J Neurochem. 2002;80:488-500.
• ¹⁴²
  Moreno-Martet M, Mestre L, Loria F, Guaza C, Fernandez-Ruiz J, de Lago E. Identification of receptors and enzymes for endocannabinoids in NSC-34 cells: relevance for in vitro studies with cannabinoids in motor neuron diseases. Neurosci Lett. 2012;508:67-72.
• ¹⁴³
  Prause J, Goswami A, Katona I, Roos A, Schnizler M, Bushuven E, et al. Altered localization, abnormal modification and loss of function of Sigma receptor-1 in amyotrophic lateral sclerosis. Hum Mol Genet. 2013;22:1581-600.
• ¹⁴⁴
  Pokrishevsky E, Grad LI, Yousefi M, Wang J, Mackenzie IR, Cashman NR. Aberrant localization of FUS and TDP43 is associated with misfolding of SOD1 in amyotrophic lateral sclerosis. PLoS One. 2012;7:e35050.
• ¹⁴⁵
  Wada T, Goparaju SK, Tooi N, Inoue H, Takahashi R, Nakatsuji N, et al. Amyotrophic lateral sclerosis model derived from human embryonic stem cells overexpressing mutant superoxide dismutase 1. Stem Cells Transl Med. 2012;1:396-402.
• ¹⁴⁶
  Ilieva H, Polymenidou M, Cleveland DW. Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J Cell Biol. 2009;187:761-72.
• ¹⁴⁷
  Liu Y, Hao W, Dawson A, Liu S, Fassbender K. Expression of amyotrophic lateral sclerosis-linked SOD1 mutant increases the neurotoxic potential of microglia via TLR2. J Biol Chem. 2009;284:3691-9.
• ¹⁴⁸
  Roberts K, Zeineddine R, Corcoran L, Li W, Campbell IL, Yerbury JJ. Extracellular aggregated Cu/Zn superoxide dismutase activates microglia to give a cytotoxic phenotype. Glia. 2013;61:409-19.
• ¹⁴⁹
  Sargsyan SA, Blackburn DJ, Barber SC, Grosskreutz J, De Vos KJ, Monk PN, et al. A comparison of in vitro properties of resting SOD1 transgenic microglia reveals evidence of reduced neuroprotective function. BMC Neurosci. 2011;12:91.
• ¹⁵⁰
  Sargsyan SA, Blackburn DJ, Barber SC, Monk PN, Shaw PJ. Mutant SOD1 G93A microglia have an inflammatory phenotype and elevated production of MCP-1. Neuroreport. 2009;20:1450-5.
• ¹⁵¹
  Weydt P, Yuen EC, Ransom BR, Moller T. Increased cytotoxic potential of microglia from ALS-transgenic mice. Glia. 2004;48:179-82.
• ¹⁵²
  Xiao Q, Zhao W, Beers DR, Yen AA, Xie W, Henkel JS, et al. Mutant SOD1(G93A) microglia are more neurotoxic relative to wild-type microglia. J Neurochem. 2007;102:2008-19.
• ¹⁵³
  Bilsland LG, Nirmalananthan N, Yip J, Greensmith L, Duchen MR. Expression of mutant SOD1 in astrocytes induces functional deficits in motoneuron mitochondria. J Neurochem. 2008;107:1271-83.
• ¹⁵⁴
  Ferraiuolo L, Higginbottom A, Heath PR, Barber S, Greenald D, Kirby J, et al. Dysregulation of astrocyte-motoneuron cross-talk in mutant superoxide dismutase 1-related amyotrophic lateral sclerosis. Brain. 2011;134:2627-41.
• ¹⁵⁵
  Ilieva EV, Ayala V, Jove M, Dalfo E, Cacabelos D, Povedano M, et al. Oxidative and endoplasmic reticulum stress interplay in sporadic amyotrophic lateral sclerosis. Brain. 2007;130:3111-23.
• ¹⁵⁶
  Park HW, Cho JS, Park CK, Jung SJ, Park CH, Lee SJ, et al. Directed induction of functional motor neuron-like cells from genetically engineered human mesenchymal stem cells. PLoS One. 2012;7:e35244.
• ¹⁵⁷
  Rothstein JD, Bristol LA, Hosler B, Brown RH, Jr., Kuncl RW. Chronic inhibition of superoxide dismutase produces apoptotic death of spinal neurons. Proc Natl Acad Sci U S A. 1994;91:4155-9.
• ¹⁵⁸
  Rothstein JD, Jin L, Dykes-Hoberg M, Kuncl RW. Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity. Proc Natl Acad Sci U S A. 1993;90:6591-5.

Publication Dates

• Publication in this collection
  2013