Human Sporadic Amyotrophic Lateral Sclerosis Research Articles

Expression and Cell Type-specific Localization of Inflammasome Sensors in the Spinal Cord of SOD1(G93A) Mice and Sporadic Amyotrophic lateral sclerosis Patients

Inflammasomes are key components of the innate immune system and activation of these multiprotein platforms is a crucial event in the etiopathology of amyotrophic lateral sclerosis (ALS). Inflammasomes consist of a pattern recognition receptor (PRR), the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and caspase 1. Exogenous or endogenous “danger signals” can trigger inflammasome assembly and promote maturation and release of pro-inflammatory cytokines, including interleukin 1β. Previous studies have demonstrated presence and activation of NLRP3 in spinal cord tissue from SOD1(G93A) mice and human sporadic ALS (sALS) patients. However, regulation and cell type-specific localization of other well-known PRRs has not yet been analysed in ALS. Here, we explored gene expression, protein concentration and cell type-specific localization of the NLRP1, NLRC4 and AIM2 inflammasomes in spinal cord samples from SOD1(G93A) mice and sALS patients. Transcription levels of NLRP1 and NLRC4, but not AIM2, were elevated in symptomatic SOD1(G93A) animals. Immunoblotting revealed elevated protein levels of NLRC4, which were significantly increased in sALS vs. control patients. Immunofluorescence studies revealed neuronal labelling of all investigated PRRs. Staining of AIM2 was detected in all types of glia, whereas glial type-specific labelling was observed for NLRP1 and NLRC4. Our findings revealed pathology-related and cell type-specific differences in the expression of subsets of PRRs. Besides NLRP3, NLRC4 appears to be linked more closely to ALS pathogenesis.

Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis.

Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

Intron retention and nuclear loss of SFPQ are molecular hallmarks of ALS

Mutations causing amyotrophic lateral sclerosis (ALS) strongly implicate ubiquitously expressed regulators of RNA processing. To understand the molecular impact of ALS-causing mutations on neuronal development and disease, we analysed transcriptomes during in vitro differentiation of motor neurons (MNs) from human control and patient-specific VCP mutant induced-pluripotent stem cells (iPSCs). We identify increased intron retention (IR) as a dominant feature of the splicing programme during early neural differentiation. Importantly, IR occurs prematurely in VCP mutant cultures compared with control counterparts. These aberrant IR events are also seen in independent RNAseq data sets from SOD1- and FUS-mutant MNs. The most significant IR is seen in the SFPQ transcript. The SFPQ protein binds extensively to its retained intron, exhibits lower nuclear abundance in VCP mutant cultures and is lost from nuclei of MNs in mouse models and human sporadic ALS. Collectively, we demonstrate SFPQ IR and nuclear loss as molecular hallmarks of familial and sporadic ALS.

Erratum to: Administration of 17β-Estradiol Improves Motoneuron Survival and Down-regulates Inflammasome Activation in Male SOD1(G93A) ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease manifested by the progressive loss of upper and lower motoneurons. The pathomechanism of ALS is complex and not yet fully understood. Neuroinflammation is believed to significantly contribute to disease progression. Inflammasome activation was recently shown in the spinal cord of human sporadic ALS patients and in the SOD1(G93A) mouse model for ALS. In the present study, we investigated the neuroprotective and anti-inflammatory effects of 17β-estradiol (E2) treatment in pre-symptomatic and symptomatic male SOD1(G93A) mice. Symptomatic mice with E2 substitution exhibited improved motor performance correlating with an increased survival of motoneurons in the lumbar spinal cord. Expression of NLRP3 inflammasome proteins and levels of activated caspase 1 and mature interleukin 1 beta were significantly reduced in SOD1(G93A) mice supplemented with E2.

Administration of 17β-Estradiol Improves Motoneuron Survival and Down-regulates Inflammasome Activation in Male SOD1(G93A) ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease manifested by the progressive loss of upper and lower motoneurons. The pathomechanism of ALS is complex and not yet fully understood. Neuroinflammation is believed to significantly contribute to disease progression. Inflammasome activation was recently shown in the spinal cord of human sporadic ALS patients and in the SOD1(G93A) mouse model for ALS. In the present study, we investigated the neuroprotective and anti-inflammatory effects of 17β-estradiol (E2) treatment in pre-symptomatic and symptomatic male SOD1(G93A) mice. Symptomatic mice with E2 substitution exhibited improved motor performance correlating with an increased survival of motoneurons in the lumbar spinal cord. Expression of NLRP3 inflammasome proteins and levels of activated caspase 1 and mature interleukin 1 beta were significantly reduced in SOD1(G93A) mice supplemented with E2.

Presymptomatic activation of the PDGF-CC pathway accelerates onset of ALS neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with unknown origins. Neurodegeneration in ALS mouse models occurs together with signs of disrupted blood-spinal cord barrier (BSCB) and regressed capillary network, but the molecular pathways contributing to these vascular pathologies remain unknown. We show that motor neurons of human sporadic ALS patients (n=12) have increased gene expression of PDGFC and its activator PLAT and presymptomatic activation of the PDGF-CC pathway in SOD1 (G93A) mice leads to BSCB dysfunction. Decrease of Pdgfc expression in SOD1 (G93A) mice restored vascular barrier properties, reduced motor neuron loss and delayed symptom onset by up to 3weeks. Similarly, lower expression levels of PDGFC or PLAT in motor neurons of sporadic ALS patients were correlated with older age at disease onset. PDGF-CC inhibition and restoration of BSCB integrity did not prevent capillary regression at disease end stage. Lower vessel density was found in spinal cords of sporadic ALS patients and the degree of regression in SOD1 (G93A) mice correlated with more aggressive progression after onset regardless of BSCB status. We conclude that PDGF-CC-induced BSCB dysfunction can contribute to timing of ALS onset, allow insight into disease origins and development of targeted novel therapies.

NLRP3 inflammasome is expressed by astrocytes in the SOD1 mouse model of ALS and in human sporadic ALS patients.

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motoneurons in the cerebral cortex, brainstem and spinal cord. Neuroinflammation plays an important role in the pathogenesis of ALS and involves the activation of microglia and astrocytes. Intracellular inflammasome complexes are part of the innate immunity as they sense and execute host inflammatory responses. The best characterized component is the NLRP3 inflammasome comprised of the NLR protein NLRP3, the adaptor ASC and pro-caspase 1. The NLRP3 inflammasome is critical for the activation of caspase 1 and the processing and release of IL1β and IL18. In this study, we investigated the expression, activation and co-localization of the NLRP3 inflammasome in the spinal cord of male SOD1(G93A) mice carrying a mutant human superoxide dismutase 1 (SOD1) variant and regarded as an animal model for ALS as well as in post-mortem tissue of ALS patients. NLRP3 and its molecular components as well as IL1β were already detectable in SOD1 mice at a pre-symptomatic stage after 9 weeks and further increased in 14 week old animals. Spinal cord astrocytes were identified as the major cell type expressing NLRP3 components. In human ALS tissue, we also found increased NLRP3, ASC, IL18 and active caspase 1 levels compared to control patients. Our findings suggest that astroglial NLRP3 inflammasome complexes are critically involved in neuroinflammation in ALS.

Decreased mRNA Expression of PGC-1α and PGC-1α-Regulated Factors in the SOD1G93AALS Mouse Model and in Human Sporadic ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss. Although the cause of ALS is unknown, oxidative stress, inflammation, and mitochondrial dysfunction have been identified as important components of its pathogenesis. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) plays a central role in the regulation of mitochondrial metabolism and biogenesis via activation of transcription factors, such as nuclear respiratory factors 1 and 2 and mitochondrial transcription factor A (Tfam). Alterations in PGC-1α expression and function have previously been described in models of Huntington and Alzheimer diseases. Moreover, the protective effects of PGC-1α have been shown in animal models of ALS. Levels of PGC-1α correlate with the number of acetylcholine receptor clusters in muscle. This is of particular interest because neurodegeneration in ALS may be a dying-back process. We investigated mRNA and protein expressions of PGC-1α and PGC-1α-regulated factors in the spinal cord and muscle tissues of SOD1 ALS mice and in ALS patients. We detected significant alterations in mRNA expression of PGC-1α and downstream factors with their earliest occurrence in muscle tissue. Our data provide evidence for a role of PGC-1α in mitochondrial dysfunction both in the ALS mouse model and in human sporadic ALS that is probably most relevant in the skeletal muscle.

Markedly reduced axonal potassium channel expression in human sporadic amyotrophic lateral sclerosis: An immunohistochemical study

Fasciculations are characteristic features of amyotrophic lateral sclerosis (ALS), suggesting abnormally increased excitability of motor axons. Previous nerve excitability studies have shown reduced axonal potassium currents in ALS patients that may contribute to the hyperexcitability and thereby generation of fasciculations. To clarify changes in axonal ion channel expression in motor axons of ALS, we performed immunohistochemistry of potassium and sodium channels in the C7 and L5 ventral/dorsal roots obtained from five autopsy cases of sporadic ALS. Compared to controls, the immunoreactivity of potassium channels (Kv1.2) was markedly reduced in the ventral roots, but normal in the dorsal roots of all the ALS patients. Nodal sodium channel expression was not significantly different in ALS patients and control subjects. Our results show prominently reduced expression of axonal potassium channels, and provide the neuropathological and biological basis for decreased accommodative potassium currents in motor axons of ALS patients. The axonal hyperexcitability would lead to generation of fasciculations, and possibly enhances motor neuron death in ALS.

Stress signaling from the endoplasmic reticulum: A central player in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the misfolding and aggregation of distinct proteins in affected tissues, however, the pathogenic cause of disease remains unknown. Recent evidence indicates that endoplasmic reticulum (ER) stress plays a central role in ALS pathogenesis. ER stress activates the unfolded protein response (UPR), a homeostatic response to misfolded proteins. The UPR is initially protective by up-regulation of specific ER stress-regulated genes and inhibition of general protein translation. However, long-term ER stress leads to cell death via apoptotic signaling, thus providing a link to neurodegeneration. Activation of the UPR is one of the earliest events in affected motor neurons of transgenic rodent models expressing ALS-linked mutant superoxide dismutase 1 (SOD1). Recently, genetic manipulation of ER stress in several different SOD1 mouse models was shown to alter disease onset and progression, implicating an active role for the UPR in disease mechanisms. Furthermore, mutations to vesicle-associated membrane protein-associated protein B (VAPB), an ER transmembrane protein involved in ER stress regulation, also cause some cases of familial ALS. ER stress also occurs in spinal cord tissues of human sporadic ALS patients, and recent evidence suggests that perturbation of the ER could occur in ALS cases associated with TAR DNA binding protein 43 (TDP-43), fused in sarcoma (FUS) and valosin containing protein (VCP). Together these findings implicate ER stress as a potential upstream mechanism involved in both familial and sporadic forms of ALS.

Is the spinal cord motoneuron exclusively a target in ALS? Comparison between astroglial reactivity in a rat model of familial ALS and in human sporadic ALS cases

Objective: Motoneurons are the focus of most investigations of amyotrophic lateral sclerosis (ALS), while the astrocyte reaction is regarded as a phenomenon secondary to neuron degeneration. Since astroglial reactivity differed in different studies of human and animal ALS models and often varied from case to case, we examined and compared astrocyte reactivity within the anterior horns of the spinal cord in a transgenic rat model of familial ALS and in human sporadic ALS (sALS) cases.Methods: Routine histological staining and immunohistochemical reactions to cytoskeletal proteins [neurofilaments, glial fibrillary acidic protein (GFAP), vimentin and tau] and proliferative markers (proliferating cell nuclear antigen and Ki-67).Results: In human sALS cases and in rats at the early pre-symptomatic and symptomatic stages of the disease, the astroglial reaction was very weak, although there was visible evidence of the morphological manifestations of motoneuron degeneration. Poor immunoreactivity to the GFAP and vimentin antigens and increased expression of tau protein were observed in astrocytes, particularly in the rat model. The astrocyte reaction was evident during a short 'transient' phase of the disease in animals, between the asymptomatic and paretic stages. Proliferating cell nuclear antigen immunoreactivity in glial and neuronal nuclei was observed only in animal material.Conclusions: Abnormalities in astrocyte cytoskeletal proteins are characteristic features for ALS, both in acquired and congenital forms of the disease. The cytoskeletal aberrations may lead to astroglial dysfunction and disturbances in glutamate uptake that may in turn increase the degeneration of motoneurons.

Integrative gene–tissue microarray-based approach for identification of human disease biomarkers: application to amyotrophic lateral sclerosis

Advances in genomics and proteomics permit rapid identification of disease-relevant genes and proteins. Challenges include biological differences between animal models and human diseases, high discordance between DNA and protein expression data and a lack of experimental models to study human complex diseases. To overcome some of these limitations, we developed an integrative approach using animal models, postmortem human material and a combination of high-throughput microarray methods to identify novel molecular markers of amyotrophic lateral sclerosis (ALS). We used laser capture microdissection coupled with microarrays to identify early transcriptome changes occurring in spinal cord motor neurons or surrounding glial cells. Two models of familial motor neuron disease, SOD1(G93A) and TAU(P301L), transgenic mice were used at the presymptomatic stage. Identified gene expression changes were predominantly model-specific. However, several genes were regulated in both models. The relevance of identified genes as clinical biomarkers was tested in the peripheral blood transcriptome of presymptomatic SOD1(G93A) animals using custom-designed ALS microarray. To confirm the relevance of identified genes in human sporadic ALS (SALS), selected corresponding protein products were examined by high-throughput immunoassays using tissue microarrays constructed from human postmortem spinal cord tissues. Genes that were identified by these experiments and located within a linkage region associated with familial ALS/frontotemporal dementia were sequenced in several families. This large-scale gene and protein expression study pointing to distinct molecular mechanisms of TAU- and SOD1-induced motor neuron degeneration identified several new SALS-relevant proteins (CNGA3, CRB1, OTUB2, MMP14, SLK, DDX58, RSPO2) and putative blood biomarkers, including Nefh, Prph and Mgll.

Protein disulphide isomerase protects against protein aggregation and is S-nitrosylated in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a rapidly progressing fatal neurodegenerative disease characterized by the presence of protein inclusions within affected motor neurons. Endoplasmic reticulum stress leading to apoptosis was recently recognized to be an important process in the pathogenesis of sporadic human amyotrophic lateral sclerosis as well as in transgenic models of mutant superoxide dismutase 1-linked familial amyotrophic lateral sclerosis. Endoplasmic reticulum stress occurs early in disease, indicating a critical role in pathogenesis, and involves upregulation of an important endoplasmic reticulum chaperone, protein disulphide isomerase. We aimed to investigate the involvement of protein disulphide isomerase in endoplasmic reticulum stress induction, protein aggregation, inclusion formation and toxicity in amyotrophic lateral sclerosis. Motor neuron-like NSC-34 cell lines were transfected with superoxide dismutase 1 and protein disulphide isomerase encoding vectors and small interfering RNA, and examined by immunocytochemistry and immunoblotting. Expression of mutant superoxide dismutase 1 induced endoplasmic reticulum stress, predominantly in cells bearing mutant superoxide dismutase 1 inclusions but also in a proportion of cells expressing mutant superoxide dismutase 1 without visible inclusions. Over-expression of protein disulphide isomerase decreased mutant superoxide dismutase 1 aggregation, inclusion formation, endoplasmic reticulum stress induction and toxicity, whereas small interfering RNA targeting protein disulphide isomerase increased mutant superoxide dismutase 1 inclusion formation, indicating a protective role for protein disulphide isomerase against superoxide dismutase 1 misfolding. Aberrant modification of protein disulphide isomerase by S-nitrosylation of active site cysteine residues has previously been shown as an important process in neurodegeneration in Parkinson's and Alzheimer's disease brain tissue, but has not been described in amyotrophic lateral sclerosis. Using a biotin switch assay, we detected increased levels of S-nitrosylated protein disulphide isomerase in transgenic mutant superoxide dismutase 1 mouse and human sporadic amyotrophic lateral sclerosis spinal cord tissues. Hence, despite upregulation, protein disulphide isomerase is also functionally inactivated in amyotrophic lateral sclerosis, which may prevent its normal protective function and contribute to disease. We also found that a small molecule mimic of the protein disulphide isomerase active site, (+/-)-trans-1,2-bis(mercaptoacetamido)cyclohexane, protected against mutant superoxide dismutase 1 inclusion formation. These studies reveal that endoplasmic reticulum stress is important in the formation of mutant superoxide dismutase 1 inclusions, and protein disulphide isomerase has an important function in ameliorating mutant superoxide dismutase 1 aggregation and toxicity. Functional inhibition of protein disulphide isomerase by S-nitrosylation may contribute to pathophysiology in both mutant superoxide dismutase 1-linked disease and sporadic amyotrophic lateral sclerosis. Protein disulphide isomerase is therefore a novel potential therapeutic target in amyotrophic lateral sclerosis and (+/-)-trans-1,2-bis(mercaptoacetamido)cyclohexane and other molecular mimics of protein disulphide isomerase could be of benefit in amyotrophic lateral sclerosis and other neurodegenerative diseases related to protein misfolding.

Involvement of CHOP, an ER-stress apoptotic mediator, in both human sporadic ALS and ALS model mice

Endoplasmic reticulum (ER) stress-induced neuronal death may play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, whether CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP), an ER-stress apoptotic mediator, is involved in the pathogenesis of ALS is controversial. Here we demonstrate the expression levels and localization of CHOP in spinal cords of both sporadic ALS patients and ALS transgenic mice by immunohistochemistry. In the spinal cords of sporadic ALS patients, CHOP was markedly up-regulated but typically expressed at low levels in those of the control. Likewise, CHOP expression increased at 14 (symptomatic stage) and 18 to 20 weeks (end stage) in ALS transgenic mice spinal cords. Furthermore, localizations of CHOP were merged in motor neurons and glial cells, such as oligodendrocytes, astrocytes, and microglia. These results indicate that the up-regulation of CHOP in motor neurons and glial cells may play pivotal roles in the pathogenesis of ALS.

シンポジウム２‐１ ＡＬＳ治療法開発の将来 孤発性ＡＬＳ疾患モデルによる病態解明と治療法開発

The mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain poorly understood even now 140 years after the first description of the disease in 1869 by Jean-Martin Charcot. Exploration of pathogenesis of ALS has long been dependent on transgenic animal models with mutations in the copper/ zinc superoxide dismutase 1 (SOD1) gene. However, the lack of therapeutic concordance between these animal models and human sporadic ALS patients is troubling. The reasons include that there might exist the differences of pathogenesis between sporadic and familial ALS and/or the disease models for sporadic ALS have not been established. We have been working on screening motor neuron-specific genes critical for pathogenesis of sporadic ALS using cDNA microarray and laser capture microdissection techniques. Many of the resultant genes are of intense interest and may provide a powerful tool for determining the molecular mechanisms of sporadic ALS. In particular, dynactin-1, a major component of dynein/dynactin complex and several cell cycle-related genes are the targets of our research. Development and analysis of new disease models for sporadic ALS based on these genes will open an avenue for novel therapeutics.

TDP-43 expression in mouse models of amyotrophic lateral sclerosis and spinal muscular atrophy

BackgroundRedistribution of nuclear TAR DNA binding protein 43 (TDP-43) to the cytoplasm and ubiquitinated inclusions of spinal motor neurons and glial cells is characteristic of amyotrophic lateral sclerosis (ALS) pathology. Recent evidence suggests that TDP-43 pathology is common to sporadic ALS and familial ALS without SOD1 mutation, but not SOD1-related fALS cases. Furthermore, it remains unclear whether TDP-43 abnormalities occur in non-ALS forms of motor neuron disease. Here, we characterise TDP-43 localisation, expression levels and post-translational modifications in mouse models of ALS and spinal muscular atrophy (SMA).ResultsTDP-43 mislocalisation to ubiquitinated inclusions or cytoplasm was notably lacking in anterior horn cells from transgenic mutant SOD1G93A mice. In addition, abnormally phosphorylated or truncated TDP-43 species were not detected in fractionated ALS mouse spinal cord or brain. Despite partial colocalisation of TDP-43 with SMN, depletion of SMN- and coilin-positive Cajal bodies in motor neurons of affected SMA mice did not alter nuclear TDP-43 distribution, expression or biochemistry in spinal cords.ConclusionThese results emphasise that TDP-43 pathology characteristic of human sporadic ALS is not a core component of the neurodegenerative mechanisms caused by SOD1 mutation or SMN deficiency in mouse models of ALS and SMA, respectively.

RETRACTED: Endoplasmic reticulum stress and induction of the unfolded protein response in human sporadic amyotrophic lateral sclerosis

The unfolded protein response (UPR) is induced at symptom onset and disease end stage in rodent models of familial amyotrophic lateral sclerosis (ALS) that express superoxide dismutase (SOD1) mutations. However, ninety percent of human ALS is sporadic and mutations in SOD1 account for only 2% of total ALS. Here we show that a full UPR, including induction of stress sensor kinases, chaperones and apoptotic mediators, is also present in spinal cords of human patients with sporadic disease. Furthermore, the UPR chaperone protein disulphide isomerase (PDI) was present in CSF and was aggregated and widely distributed throughout the motor neurons of these patients. We also show up-regulation of UPR prior to the onset of symptoms in SOD1 rodents, implying an active role in disease. This study offers new insights into pathogenesis, placing ER stress onto a generic pathophysiology for ALS.

Muscular mitochondrial function in amyotrophic lateral sclerosis is progressively altered as the disease develops: A temporal study in man

We performed repeated analysis of mitochondrial respiratory function in skeletal muscle (SM) of patients with early-stage sporadic amyotrophic lateral sclerosis (SALS) to determine whether mitochondrial function was altered as the disease advanced. SM biopsies were obtained from 7 patients with newly diagnosed SALS, the same 7 patients 3 months later, and 7 sedentary controls. Muscle fibers were permeabilized with saponin, then skinned and placed in an oxygraphic chamber to measure basal and maximal adenosine diphosphate (ADP)-stimulated respiration rates and to assess mitochondrial regulation by ADP. We found that the maximal oxidative phosphorylation capacity of muscular mitochondria significantly increased, and muscular mitochondrial respiratory complex IV activity significantly decreased as the disease advanced. This temporal study demonstrates for the first time that mitochondrial function in SM in human SALS is progressively altered as the disease develops.

Up-regulation of mitochondrial uncoupling protein 3 reveals an early muscular metabolic defect in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting primarily motor neurons. Growing evidence suggests a mitochondrial defect in ALS. The precise molecular mechanisms underlying those defects are unknown. We studied the expression of mitochondrial uncoupling proteins (UCPs), key regulators of mitochondrial functions, in tissues from a mouse model of ALS (SOD1 G86R transgenic mice) and from muscular biopsies of human sporadic ALS. Surprisingly, in SOD1 G86R mice, UCPs, and particularly UCP3, were upregulated in skeletal muscle but not in spinal cord. Consistent with this pattern of expression, ATP levels were selectively depleted in muscle but not in neural tissues 1 month before disease onset and the respiratory control ratio of isolated mitochondria is decreased. UCP3 up-regulation was not observed in experimentally denervated muscles, suggesting that changes in muscular UCP3 expression are associated with the physiopathological processes of ALS. This is further supported by our observation of increased UCP3 levels in human ALS muscular biopsies. We propose that UCP3 up-regulation in skeletal muscle contributes to the characteristic mitochondrial damage of ALS and to the onset of the disease. Moreover, since skeletal muscle is a key metabolic tissue, our findings suggest that ALS may not solely arise from neuronal events but also from more generalized metabolic defects.

Detection by 4-parameter microscopic imaging and increase of rare mononuclear blood leukocyte types expressing the FcγRIII receptor (CD 16) for immunoglobulin G in human sporadic amyotrophic lateral sclerosis (ALS)

By using quantitative multiparameter microscopic imaging we demonstrate concentration of two peripheral mononuclear blood leukocyte types expressing the FcγRIII receptor for immunogobulin G in a clinical subgroup of amyotrophic lateral sclerosis (ALS, n = 9) showing bulbar palsy (ALS BP) and/or predominant involvement of the upper motor neuron (ALS C). Triple fluorescence staining and overlay with phase contrast images (4 parameters) reveals that cell type 1 co-expresses FcγRIII (CD16), CD8 and CD57 surface antigens (ALS C) 50 ± 33.6 cells/μl, P = 0.0012; ALS BP 16.5 ± 32.4, P = 0.029). This cell type is not observed in healthy individuals ( n = 8) and is only insignificantly increased ( P > 0.05) in neurological disease controls (stroke, n = 3, 2.1 ± 3.7; polymyositis, n = 6, 1.5 ± 4.0 cells/μl) and in ALS cases with peripheral symptoms (ALS P n = 12: 7.6 ± 8.7). Cell type 2 co-expresses FcγRIII (CD16) and CD8, but is negative for CD57 (ALS C 60.1 ± 19.3; ALS BP 24.2 ± 28.0 cells/μl). These findings are consistent with previous reports on IgG isotype changes and immune-cell invasion of the motor system in ALS.