Applications of Arrays to Neurological Disorders

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Initial gene expression studies in neurology focused on using tissue homo-genates from affected regions of the brain to gain further insights into the pathogenic mechanisms responsible for neuronal cell death. More recently, the application of LCM and linear amplification has interrogated the gene expression of the vulnerable neuronal cell type, without the dilution effect from supporting cells of the central nervous system (CNS). Illustrative examples from Huntington's disease (HD), Alzheimer's disease (AD), multiple sclerosis (MS), and motor neuron disease (MND) demonstrate how micro-array technology has been used to advance the knowledge of these diseases.

5.1. Huntington's Disease

HD is a midlife onset, progressive neurodegenerative disease characterized by motor impairment, cognitive decline, and psychiatric symptoms. Histo-pathologically, the earliest and most severe neurodegeneration occurs in the GABAergic cells, or medium spiny neurons of the caudate nucleus, although atrophy is also seen in the putamen, globus pallidus, and cerebral cortex [68]. For this reason, gene expression studies have focused on these regions of the brain. In 1993, the genetic cause of the disease was identified as an expanded repeat of the CAG codon encoding the polyglutamine tract, in exon 1 of the IT15, or HD gene [69]. The encoded protein, huntingtin (HTT), is ubiqui-tiously expressed throughout the brain and body, suggesting a selective vulnerability in the neurons affected.

Many studies have been carried out on HD mouse models and human material to investigate the basic mechanisms of neurodegeneration of mutant HTT protein [70-72], the effects of mutant HTT protein length [73], the variability in expression changes in different regions of the brain [74], the effects of potential HD drug treatments [13, 75, 76], and to identify potential biomarkers which correlate with the progression of the disease [77]. The next sections will discuss each of these applications in turn.

5.1.1. Gene Expression Studies in HD Mouse Models

To provide an insight into the pathways involved in HD neurodegeneration, gene expression studies of the striatum were performed on the mouse model of HD, R6/2, which expresses exon 1 of the HD gene with 140-150 CAG repeats under control of the HD promoter [70]. Eighty percent of cells in the mouse striatum were estimated to consist of medium spiny GABAergic neurons. Decreased neurotransmitter receptor gene expression was identified in the striata of transgenic mice. In addition, decreases were seen in other components of neuronal signaling pathways (specifically those of dopamine and glutamate), and in genes involved in calcium signaling pathways, ion channels, transcription, metabolism, and cell structure, while increases were seen in inflammation and cell cycle genes. Interestingly, differential gene expression was seen early in the disease, at 6 weeks, when only subtle motor defects were evident, suggesting an accumulation of neuronal damage underlies the progressive nature of the disease.

To determine the extent of gene expression changes responding specifically to mutant HTT, rather than a polyglutamine repeat, further gene expression studies were conducted using a mouse model of dentatorubral-pallidoluysian atrophy (DRPLA) [71]. This is also an autosomal dominant neurodegenerative disease, caused by expansion of 49-88 CAG repeats in the atrophin-1 gene (Atn1). The At-65Q mice contained an expansion of 65 repeats in Atn1 under control of the same promoter as the Htt gene in the N171-82Q mice. These mice express a peptide consisting of 171 amino acids of the N-terminus of HTT, with 82 CAG repeats. Analysis of cerebellar gene expression profiles in the two models compared to wild-type litter mates identified 184 genes in common, suggesting these are altered in response to an expanded polygluta-mine, rather than representing gene expression changes specific to the mutant protein. In addition, 74 of these changes were also seen in the R6/2 HD mice [72], and four genes, including enkephalin, were also confirmed as altered in the cerebellum from mouse models of two other CAG repeat disorders, spinal cerebellar ataxia 7 and spinal bulbar muscular atrophy [71]. However, these changes were not seen in transgenic mice expressing a full-length HTT protein with 72 CAG repeats.

To determine the extent of the effect of HTT protein length on gene expression, studies have also been conducted comparing mouse models expressing the short truncated HTT protein with expanded repeats (R6/2 and N171-82Q mice) with a larger truncated HTT protein (HD46 and HD100 containing 46 and 100 CAG repeats along with the first 964 amino acids) and full-length mutant HTT protein (YAC72 containing 72 CAG repeats) [73]. These results indicated that striata expressing the longer HTT proteins showed fewer expression changes compared to those with the shorter truncated protein at pathologically equivalent ages. In addition, previously identified gene expression changes in the R6/2 and N171-82Q mice were not among those genes differentially expressed in mice transgenic for the longer mutant HTT proteins. However, it should be noted that shorter, proteolytically cleaved HTT is present in both HD and control brain tissue [78], and with changes in the R6/2 mice also present in human HD brain [70], it has been proposed that mutant HTT has an effect following proteolysis of the complete protein into the small N-terminal fragments.

5.1.2. Gene Expression Studies in HD Human HD Cases

The most comprehensive study to date of human postmortem HD tissue analyzed gene expression from four brain regions in 44 HD cases and 36 controls [74]. Gene expression profiles were obtained from the caudate nucleus, which shows the most severe and earliest pathology, cerebellum which shows little pathology, motor cortex which controls the motor function that is altered early in the disease, and prefrontal association cortex, involved in cognitive processing which deteriorates later in the disease. Twenty-one percent of the genes interrogated were differentially expressed in the HD caudate samples, compared to 3% in the HD motor cortex and 1% in the HD cerebellum, while prefrontal cortex showed no changes other than those expected by chance. The HD caudate samples also showed the largest fold changes in expression. Interestingly, of the genes in the motor cortex also altered in the caudate, the majority were altered in the same direction, suggesting similar changes occur in different brain regions as well as changes specific to that cell type. To address the issue of whether the large number of gene changes in the caudate was due to cell loss of the medium spiny neurons, LCM was used to isolate the same number of neurons from both HD cases and controls. Subsequent microarray analysis confirmed 77% of the decreased genes and 65% of the increased genes were concordant with those genes differentially expressed in the whole caudate samples. As expected, the largest number of gene expression changes was in the category of neuronal signaling and homeostasis, specifically in the neurotransporter receptors. In addition, genes involved in intracellular signaling, and proton and metal ion transport were also implicated. Thus, this global gene expression study confirms and extends the genes differentially expressed that occur in the presence of the mutant HTT protein, while demonstrating the both similarities and differences in regional expression changes that occur in the HD brain.

Mutant HTT has been found to bind to other polyglutamine containing proteins, including transcription factors [79], and forms aggregates, with sequestration of other cellular proteins. Thus, the proposed mechanism of action for mutant HTT is through aberrant protein-protein interactions, particularly involving transcriptional dysregulation, as demonstrated by the human and mouse microarray studies [71, 72, 74]. This subsequent disruption of transcription is thought to underlie the apparent involvement of oxidative stress, mitochondrial dysfunction, apoptosis, energy metabolism disturbances, and excitotoxicity in neuronal cell death.

5.1.3. Transcriptional Effects of Drug Treatments in HD

The dysregulation of transcription has provided a target for therapeutic intervention, and microarray analysis has been used to follow the effect of the drug in the treated samples. For example, acetylation of histones regulates the gene expression of between 2 and 5% of genes [80], allowing transcription factors access to regions of DNA which are tightly packed in the chromatin. While histone acetyltransferases add the acetyl group onto the lysine residues found in the histones N-terminal tails, the histone deacetylases (HDAC) remove them, in a highly dynamic process. Thus, gene transcription occurs when the rate of acetylation exceeds that of deacetylation. Mutant HTT was found to bind and sequester p53- and CREB-binding protein (CBP) into aggregates of HTT, and was associated with gene repression of two p53 promoters [81]. CBP is a transcriptional activator and functions as a histone acetyltransferase [82]. Following the demonstration that cognitive defects observed in CBP+/— transgenic mice were ameliorated following treatment with HDAC inhibitors, HDAC inhibitiors have been trialed in mouse models of HD and the gene expression response analyzed using microarrays.

In R6/2 mice injected with an HDAC inhibitor, sodium butyrate, from 3 weeks of age, survival was extended in a dose-dependent manner by up to 20%, motor performance was enhanced and pathological improvements showed reduced atrophy of the striatum, with a 73% reduction in lesion volume [75]. Subsequent microarray studies, in mice treated for 2 weeks from 6 weeks of age, showed selective changes in the sodium butyrate-treated mice, rather than a global reversal of gene expression. It was suggested that this may be due to prior sequestration of key transcription factors into the mutant HTT aggregates. A further study looked at the treatment of a different HD mouse model, N171-82Q, with an alternative HDAC inhibitor—phenylbutyrate [13]. The aim of this study was to investigate the effects of drug treatment following symptom onset. Intraperitoneal injections from 75 days extended survival of the HD mice by 23%, and this was associated with reduced gross brain atrophy, specifically striatal neuron atrophy. Analysis of striatal gene expression on Affymetrix U74Av2 GeneChips reported 11 genes significantly increased, including glutathione S-transferase (GSTm3), proteasomal subunits (Psma3 and ATPase3), and ubiquitin-specific protease 29, and 6 genes significantly decreased by phenylbutyrate treatment, including apoptotic genes (Casp9 and Cflar). Both apoptosis and aberrant protein degradation have been demonstrated in HD, and therefore, reduction of caspase-9 expression, which was associated with a decrease in active cas-pase-3, and increases in the ubiquitin-proteasome subunits may contribute to the beneficial effects of phenylbutyrate treatment.

An alternative therapeutic strategy has focused on improving cognitive features of HD, and the effect of this treatment on gene expression has been studied [76]. R6/2 mice were treated from 5 weeks old, when they exhibit spatial learning difficulties, with a cocktail of tacrine (an acetylcholine esterase inhibitor, which results in a global increase of brain acetylcholine levels), moclobemide (an antidepressant that inhibits monoamine oxidase A and thereby prevents noradrenaline and 5-hydroxytryptamine breakdown), and creatine (a supplement which has shown to increase muscle mass, improve muscle strength in a wide range of neuromuscular disorders, as well as having a beneficial effect on mitochondrial function [83, 84]). Treated mice showed improved results in two cognitive tasks, compared to untreated mice, although they were not restored to the same level as WT mice, suggesting that the drugs act by preventing further deterioration. Gene expression analysis using U74Av2 GeneChips (Affymetrix) identified 640 differentially expressed genes of which 333 were reversed toward normal levels by the cocktail of drugs. As there were no significant improvements in motor function following treatment, these results highlight those genes specifically involved in the cognitive portion of the disease pathogenesis. The authors suggest, therefore, that combinatorial drug therapies targeting both cognitive and motor defects should be considered.

5.1.4. Identification of Biomarkers in HD

Although HD carrier status can be definitively diagnosed through genetic testing, numerous neurological disorders can only have a confirmed diagnosis at postmortem. However, HD has provided a genetically defined population in which biomarkers for the disease, and indeed biomarker changes that correlate with the progression of the disease, have been discovered in blood [77]. Previous array work demonstrated that mutant HTT, as it is ubiqui-tiously expressed, caused similar gene expression changes in nonneuronal tissue such as muscle [72], and normal and mutant HTT have been found in HD blood samples [85]. Gene profiles of peripheral blood from symptomatic patients (average 49.6 years) and late presymptomatic individuals (average 39 years) were significantly different to controls, with a large number of genes showing an increase in expression [77]. Following selection of 12 validated genes to use as biomarkers, PCA was able to distinguish the late presymptomatic cases from both controls and symptomatic patients, although the expression levels of these genes were more similar to those of symptomatic cases. Further studies in early presymptomatic individuals (average 22.5 years) demonstrated that expression of these genes increased over time, prior to symptom onset. Most importantly, analysis of blood from HD cases involved in a dose-finding study for the HDAC inhibitor sodium phenylbutyrate showed a significant decrease in the gene expression of those 12 genes following treatment. Thus, following the generation of biomarkers from microarray data, not only can the progression of the disease be monitored, but drug specificity and efficacy can be assessed, just by taking a blood sample. As such, this is an important research advance to be applied to not only other neurological disorders, but many other genetic and sporadic diseases.

5.2. Multiple Sclerosis

Ms is an autoimmune disease that attacks the myelin sheath of oligoden-drocytes around the neuronal axons. This allows the axonal cytoskeleton to be damaged, bringing about secondary axonal loss and persisting neurological dysfunction. The characteristic pathology is of a lesion or plaque in the CNS white matter, formed by inflammation and demyelination and these can be classified into active, chronic active, or chronic silent plaques [86].

The first report applying microarrays to Ms sampled gene expression in normal white matter, and compared this to acute lesions in white matter of the same patient [87]. The changes reflected altered cell metabolism and increased expression of cytokines and cell adhesion molecules characteristic of an immune response. A second study by the same group analyzed changes in gene expression from two patients, the first having both acute and chronically active lesions, while the second had chronic lesions. Comparisons were made with both control human white matter and two animal models of experimental autoimmune encephalomelitis (EAE) [88]. EAE can be induced by immunization of mice with myelin oligodendrocyte protein (MOG), proteo-lipid protein (PLP), or myelin basic protein (MBP). The resulting symptoms, produced by a targeted attack on the oligodendrocytes, and subsequent recovery following elimination of the immune cells from the CNS, are reminiscent of those in MS, complete with recurrent relapses. Only four genes were consistently upregulated in the MS lesions and both MOG and MBP induced EAE. One of the genes, arachidonate 5-lipoxygenase (5-LOX), was upregu-lated 83-fold in the acute/chronic active lesion patient and 22-fold in the chronic lesion patient, and is of particular interest as it is an important enzyme in the synthesis of the potent immune mediators, leukotrienes. Follow-up studies showed that 5-LOx knockout mice did exhibit less anxious behavior than controls [89], but contrary to expectations, given that there is an increase in 5-LOx when EAE is induced in normal mice, the 5-LOx knockout mice were more susceptible to EAE than controls [90].

Further investigation of differential gene expression in EAE induced by MOG demonstrated increases in immune response genes such as antigen receptors, MHC molecules, chemokines, and cytokines [91]. In addition, changes in genes specifically expressed in the spinal cord were investigated, and the study found those genes involved with neurogenesis and neuronal repair genes were increased, while genes encoding ion channels, neurotrans-mitters, and growth factors were decreased. Differential gene expression of this "neuronal response,'' in contrast with immune response, continues in recovering animals even after the immune response has returned to normal.

Treatment of EAE with metalloproteinase (MMP) inhibitors can prevent or reverse the progression of EAE, and microarray analysis demonstrated that MMP inhibitors reduced the expression of several genes, including osteopontin (oPN) following treatment of EAE induced in rats [92]. Subsequent studies investigated the relevance of OPN in MS, and several polymorphisms within the gene have been associated with disease susceptibility, disease progression, and age of onset, although not in all studies (reviewed by Steinman and Zamvil [93]). However, expression levels are clearly linked with the disease, with differential increases of OPN in both relapsing and remitting MS cases, and chronic active and acute plaques [94].

The three types of MS lesions have characteristic gene expression changes [95,96]. Profiling of one acute, two chronic active, and one chronic silent lesion not only identified the involvement of increased proinflammatory cytokine activity and decreased expression of genes encoding myelin-associated proteins but also showed the acute lesion to cluster separately from the chronic silent lesion, which was more similar to the white matter profile of controls [95]. Further work identified there were more differentially expressed genes in the chronic active lesions compared to the chronic silent lesions, and also the margins of the chronic active lesions showed increased transcriptional activity, particularly in genes encoding immune mediators [96].

Thus, microarrays have shown differential gene expression of markers such as OPN and 5-LOX correlate with disease state, while lesion profiling has identified a transcriptional distinction between the types of the lesions found in MS cases.

5.3. Alzheimer's Disease

AD is a neurodegenerative disease characterized by progressive cognitive decline, and is the most common cause of dementia in the elderly. First described by Alzheimer in 1907, a definite diagnosis of AD can only be made postmortem by neuropathological examination, through the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFT), neuronal loss, and synaptic dysfunction in the hippocampus and cerebral cortex. While 95% of AD is late onset (>65 years), genetic factors have been implicated in early onset AD, with mutations identified in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), or amyloid precursor protein (APP) genes. Studies on these genetically defined individuals and transgenic mice for the genes are increasing the understanding of the molecular mechanisms underlying AD, with the aim to improve the diagnosis and treatment of the disease for all cases.

5.3.1. Gene Expression Studies in AD Mouse Models

Transgenic mice have been generated carrying missense mutations in the PSEN1 gene, deletion of exon 9 (caused by a G to T substitution in the splice site acceptor site), as well as a PSEN1 knockout mouse. A study by Mirnics et al. [97] compared the gene expression profiles from hippocampus of the exon 9 deleted mouse (AE9) with those from the PSEN1 knockout mice. Interestingly, the majority of the genes in common showed contradictory directions of change, and it was suggested that this represented the effect of the "gain of function'' of the PSEN1 mutations, while the similarly changed genes may represent the loss of function effects that have also been reported [98, 99].

Amyloid precursor protein (APP) is alternatively spliced to express several isoforms [100], of which APP695 is preferentially expressed in neuronal tissue [101]. APP is cleaved first by ^-secretase, and then by 7-secretase to form ^-amyloid-40 and ^-amyloid-42. It is these ^-amyloid isoforms that aggregate in AD. PSEN1 has been demonstrated to be involved in the 7-secretase proteolytic cleavage of APP, with neuronal cultures from PSEN1-deficient mice showing reduced production of ^-amyloid-42 [102]. Microarray analysis of the Tg2576 mouse model (carrying the K670N/M671L mutations) examined the changes in gene expression that occur during the progression of the disease [103]. Transcription profiles of the cerebral cortex of mice at 2, 5, and 18 months, corresponding to long before, immediately before, and after the appearance of ^-amyloid plaques, respectively, identified mitochon-drial energy metabolism genes (particularly those related to oxidative phos-phorylation) and apoptosis genes to be upregulated at all three time points, compared to controls. Additional gene expression changes were identified in the 18-month mice, in functional pathways, including transcription, cell cycle development, and signal transduction. The finding of early mitochondrial involvement correlates with findings in the human disease from Hirai et al. [104], and the presence of this dysfunction in the mouse model now allows the exact nature of mitochondrial involvement to be determined, and also whether mitochondrial-enhancing drug treatments will be beneficial [103].

Double transgenic mice, carrying the APP (K670N/M671L) and the PSEN1 (A246E) mutations, show cognitive impairment which is consistently present by 15 months, and show this to be progressive as ^-amyloid deposition accumulates [105]. Gene expression profiling showed a decrease in genes required for normal memory function (Arc, Nur77, Zif268) and neuronal/synaptic activity (NaKATPasealII), and an increase in inflammatory-related and the acute-phase response genes (including GFAP and ApoE), reflecting glial activation, in the double-mutant mice [106]. Interestingly, most of these changes were restricted to regions of the brain showing amyloid deposition. Q-PCR of human AD brain also showed reduced expression of the memory-related genes specifically in regions affected by ^-amyloid deposits. Thus, the authors proposed that memory dysfunction occurs early, before synaptic degeneration and neuronal cell death, which is seen in human postmortem material.

5.3.2. Gene Expression Studies in Human AD Cases

One of the first studies to apply microarray technology to human postmortem AD cases used linear amplification of RNA methodology to examine the expression profile of tangle bearing CA1 neurons from AD patients compared to normal CA1 neurons in controls [107]. This demonstrated decreases in genes whose proteins had previously been implicated in AD such as those involved in the cytoskeleton, the synapse, and both glutamate and dopamine receptors. In addition, genes such as utrophin and glutaredoxin were found upregulated in the tangles and were thought to represent novel mediators of NFT formation, or neurodegeneration. Microarray analysis of AD brains has identified gene expression changes that correlate with AD markers such as Mini Mental Status Examination (MMSE) and NFT scores [108]. For example, the microfibrillary-associated protein 1 was upregulated in AD cases as NFT score increased, while the G-protein-coupled receptor 22 was decreased as the MMSE score decreased in AD cases. A further study has shown that analysis of genes expressed in fibroblasts from carriers of APP (K670N/ M671L and E693G) and PSEN1 (H163Y) mutations allows them to be clustered together separated from siblings free of the mutation, independently of the mutant gene. This suggests a similar molecular mechanism underlies the effect of these mutant genes on the cell, which correlates with PSEN1 being involved in ^-amyloid production. The expression profiles of senile plaques in the hippocampus have also been investigated, and cholinergic basal forebrain neurons, which supply the cholinergic fibers to the hippocampus and cerebral cortex (as reviewed by Ginsberg et al. [109]). These studies have identified that the senile plaques contain predominantly neuronal mRNAs, suggesting that they are formed through the accumulation of degenerating neurons [110], and that expression levels of high-affinity nerve growth factor receptors (trk) are significantly downregulated during the progression of the disease, expression correlates with declining MMSE scores, and therefore may represent an early biomarker at disease onset [111].

In a recent publication, the largest number of AD brains to date were analyzed by microarray. Unlike the previous studies, the RNA from 61 AD frontal cortex samples and 53 controls samples were pooled, and multiple hybridization experiments took place on two-array platforms. Only 3 of the top 30 differentially expressed genes from each platform were found to be in common: RGS4 and RAB3A were decreased and ITPKB was increased. However, real-time PCR with the individual samples confirmed these changes were specific to the AD patients, and these data support the evidence of disrupted intracellular calcium signaling in AD [112].

Finally, a review has compared the expression data obtained from both transgenic AD models and human postmortem AD material [113]. The human postmortem studies show more overlap in genes that are downregulated than upregulated, while the mouse studies, which include mice carrying single and double AD mutant genes, show a greater overlap with the study by Blalock et al. [108] than the other human studies. This may be explained by the inclusion of cases in the early stages of AD, rather than just advanced AD cases, and the mice models, such as the APP mutant mice, mimicking earlier gene expression changes such as changes in energy transduction, before loss of neurons. On the basis of these microarray findings, the authors proposed a model for AD progression, beginning with localized oligodendrocytes causing remyelination responses in the neuronal axons [113]. This causes secretion of growth factors from the oligodendrocytes, and compensatory tumor suppressor responses in neurons and astrocytes. This then leads to protein aggregation, abnormal axonal-myelin interactions, and NFT formation, and may also explain the observation that pathological changes in AD progress along myelinated axons.

5.4. Motor Neuron Disease

MND is a group of progressive neurodegenerative disorders encompassing the clinical subtypes of amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, primary bulbar palsy, and progressive muscular atrophy. Characterized by the cell death of upper and/or lower motor neurons in the motor cortex, brainstem, and spinal cord, the exact mechanisms of motor neuron injury are unknown, but are thought to involve oxidative stress [114], exci-totoxicity [115], protein aggregation [116], mitochondrial dysfunction [117], and genetic factors [118]. While the majority of cases are sporadic, 5-10% of cases are familial, usually with an autosomal dominant inheritance. The first gene to be identified as causative, and to date the most common, is the Cu/Zn superoxide dismutase (SOD1) gene [119]. Encoding a ubiquitously expressed free radical scavenging enzyme, mutations lead to the mutant protein possessing a toxic gain of function, thought to result from aberrant handling of free radical species and/or protein aggregation.

5.4.1. Profiling Spinal Cord Homogenates in MND

Microarray technology has been applied to determine the exact mechanisms by which mutant SOD1 causes motor neuron cell death. Due to clinical similarity of the SOD1-related MND cases to other familial and sporadic cases, further understanding of the pathogenesis in the SOD1 cases may well be applicable to MND as a whole. The first studies used lumbar spinal cord homogenates from a transgenic mouse cell line generated to express the human mutant G93A SOD1 protein [120-122]. These mice develop symptoms around 90 days, with hind limb weakness progressing to paralysis at 140-150 days. However, pathological changes occur in the motor axons as early as 30 days, with the vacuolar changes becoming evident in the motor neuron cell body by 60 days, along with Golgi fragmentation and mitochon-drial swelling. By 90 days, glial activation is present as are inclusion bodies and SOD1 protein aggregates.

These gene expression studies using the G93A SOD1 transgenic mice have also allowed the progressive nature of the disease to be studied. Fewer differentially expressed genes have been identified in the presymptomatic mice (60 days) compared to early symptomatic mice (90 days), while the majority of gene expression changes are seen at end stage of the disease (120 days) [121, 122]. In symptomatic mice, there was strong evidence of an inflammatory response from reactive astrocytes and activated microglial, as well as a potential adaptive response to metal ion dysfunction, postulated to be the result of increased intracellular iron caused by mitochondrial dysfunction. Other pathways affected included lipid metabolism [121], cytoskeletal architecture [122], and proteins involved in the differentiation/ maturation of the spinal cord population [122]. The relevance of these pathways to the human disease is supported by gene expression profiling studies of postmortem tissue from sporadic ALS cases [123-125]. Interestingly, analysis of the human postmortem material also identified significant alterations in genes encoding ubiquitin/proteasome pathway components and other protein degradation mechanisms, though whether dysregulation of protein degradation is the cause of the protein aggregations observed, or whether these are a protective measure, sequestering faulty cytosolic proteins, remains to be determined. Dangond et al. [123] also analyzed the gene expression of two familial ALS cases, one of which carried a mutation in the SOD1 gene, and found a significant number of genes which were altered specifically in these two cases, including increased expression of genes expressed in response to high calcium levels and genes involved in the cell cycle.

5.4.2. Profiling Cellular Models of MND

To determine the specific response of motor neurons to the presence of mutant SOD1, without contamination of other cell types and the effects of their interactions with motor neurons, gene expression profiling has been undertaken on the motor neuronal cell line, NSC34, transfected with mutant SOD1 [126]. This study demonstrated, in contrast to the whole tissue homogenate studies, that there was a marked degree of transcriptional repression in the presence of mutant SOD1, with reduced gene expression demonstrated in the antioxidant response pathway, and particularly the "programmed cell life'' genes under the transcriptional control of Nrf2. Additional pathways affected included genes involved in protein degradation, cell death/survival, the immune response, and the heat-shock response.

5.4.3. Profiling Motor Neurons in MND

Gene profiles from motor neurons in the spinal cord can now be determined through the combined technologies of LCM and linear amplification of RNA. A comparative study of ventral horn and isolated motor neurons in ALs cases and controls demonstrated how few motor neuron-specific gene expression changes were represented in the whole tissue homogenates from the ventral horn [127]. Consistent with the cell model findings, ALS motor neurons showed more genes were decreased (3%) than increased (1%), whereas in the ALS ventral horn samples, more genes were increased (0.7% vs 0.2% of 4845 transcripts interrogated). This can be explained by the influence of reactive gliosis and other cellular reactions in the nonneuronal cells, and through reduced motor neuron number in the ventral horn of ALs cases. Major functional groups that were found to have genes downregulated included cell receptors and intracellular signaling, transcription, metabolism, and cytoskeleton architecture, while genes encoding cell death associated proteins, secreted and extracellular communication proteins, and cell cycle regulators were all upregulated.

LCM has also been applied to isolate MN from the transgenic G93A sOD1 mice [128]. The authors demonstrate the increasing changes in gene expression that occur as the disease progresses, although, in contrast to the human and cell model studies, they show a larger number of genes increased than decreased. The majority of increased genes were involved in cell growth and/or maintenance. Only 12 genes were differentially expressed at all three time points though the progression of the disease, and one of the genes increased encodes vimentin. This is an intermediate filament involved in retrograde transport and may also play a role in neurite extension. Vimentin inclusions were seen in presymptomatic mice, and became more abundant as the disease progressed [128]. Interestingly, this increased gene expression is not specific to the sOD1 mice but also occurs in two other neurodegenerative mouse models, pmn and wobbler [129].

Gene expression studies on presymptomatic mice demonstrated vimentin to be the only gene differentially regulated in all three models, although 11 genes were differentially expressed in the pmn and wobbler mice [130], which are both autosomal recessive models of neurodegeneration arising from a spontaneous mutation event [131, 132]. These studies aimed to identify early events and responses in the neurodegenerative process, and have determined both common and gene-specific induced changes in gene expression.

In summary, gene expression studies have demonstrated the involvement of the ubiquitin/proteasome pathways, and dysregulation of mitochondria and cytoskeletal structures, supporting previous hypotheses. One of the novel findings that MN are trying to reenter cell division is surprising as in these postmitotic cells this will lead to cell death. Most importantly, these studies have demonstrated that gene expression changes of whole tissue homogenates do not portray all the differentially expressed genes in the individual cell types present.

5.5. Expression Profiling Other Neurological Disorders

In addition to the examples listed above, gene expression arrays have also been applied to Parkinson's disease (PD), identifying abnormal iron metabolism [133], oxidative stress, and protein aggregation occurring in both familial [134] and sporadic PD [135], as well as identifying genes differentially expressed in a cellular model of DJ-1-associated familial PD [136]. Further insights into Creutzfeldt-Jakob disease (CJD) have been discovered by gene expression profiling the frontal cortex, demonstrating increases in genes encoding immune and stress response, cell death, and cell cycle proteins and decreases in genes encoding synaptic proteins [137]. Importantly, although there were common pathways affected in the human CJD and the prion disease mouse models, there was a larger immune response present in the mouse, while the human CJD cases showed more apoptotic-related genes to be differentially expressed. It is crucial to be aware of such differences, when focusing on therapeutic targets identified in mouse models to slow down the progression of the human disease.

The application of microarrays to these neurological disorders, both genetic and sporadic in origin, has generated a significant amount of information regarding the molecular events occurring in both the affected regions and vulnerable cell populations. Disease progression, drug treatment, and nonneuronal samples, such as blood, can all be scrutinized at the level of transcriptional responses, generating further therapeutic targets, and identifying candidate susceptibility genes and biomarkers.

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