Variant Of Amyotrophic Lateral Sclerosis Research Articles

Multiple variants in families with amyotrophic lateral sclerosis and frontotemporal dementia related to C9orf72 repeat expansion: further observations on their oligogenic nature.

The C9orf72 repeat expansion (RE) is one of the most frequent causative mutations of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it is still unclear how the C9orf72 RE can lead to a heterogeneous phenotype. Several reports have shown the coexistence of mutations in multiple ALS/FTD causative genes in the same family, suggesting an oligogenic etiology for ALS and FTD. Our aim was to investigate this phenomenon in an Italian group of ALS/FTD pedigrees carrying the C9orf72 RE. We included 11 subjects from 11 pedigrees with ALS/FTD and the C9orf72 RE. Mutation screening of FUS, SOD1 and TARDBP genes was performed by direct sequencing. A dementia-specific custom-designed targeted next-generation sequencing panel was used for screening dementia-associated genes mutations. We found genetic variants in additional ALS or dementia-related genes in four pedigrees, including the p.V47A variant in the TYROBP gene. As a group, double mutation carriers displayed a tendency toward a younger age at onset and a higher frequency of positive familiar history and of parkinsonism. Our observation supports the hypothesis that the co-presence of mutations in different genes may be relevant for the clinical expression of ALS/FTD and of their oligogenic nature.

Whole exome sequencing and DNA methylation analysis in a clinical amyotrophic lateral sclerosis cohort.

BackgroundGene discovery has provided remarkable biological insights into amyotrophic lateral sclerosis (ALS). One challenge for clinical application of genetic testing is critical evaluation of the significance of reported variants.MethodsWe use whole exome sequencing (WES) to develop a clinically relevant approach to identify a subset of ALS patients harboring likely pathogenic mutations. In parallel, we assess if DNA methylation can be used to screen for pathogenicity of novel variants since a methylation signature has been shown to associate with the pathogenic C9orf72 expansion, but has not been explored for other ALS mutations. Australian patients identified with ALS‐relevant variants were cross‐checked with population databases and case reports to critically assess whether they were “likely causal,” “uncertain significance,” or “unlikely causal.”ResultsPublished ALS variants were identified in >10% of patients; however, in only 3% of patients (4/120) could these be confidently considered pathogenic (in SOD1 and TARDBP). We found no evidence for a differential DNA methylation signature in these mutation carriers.ConclusionsThe use of WES in a typical ALS clinic demonstrates a critical approach to variant assessment with the capability to combine cohorts to enhance the largely unknown genetic basis of ALS.

Kaplan-Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset.

Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼103 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R2 = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

The concept and diagnostic criteria of primary lateral sclerosis.

Primary lateral sclerosis (PLS) is commonly considered as a motor neuron disease (MND) variant which almost exclusively affects upper motor neurons (UMN). There is still no consensus whether PLS should be regarded as an independent disease entity separate from amyotrophic lateral sclerosis (ALS) or as a comparatively slowly progressive variant of ALS. Given these different views, clinical diagnosis of PLS is a challenge. In this multicenter study, we analyzed clinical features of patients diagnosed with PLS in four specialized MND centers. We retrospectively analyzed clinical, laboratory, imaging, and electrophysiological data of 76 patients with PLS diagnosed in four specialized ALS centers. We analyzed the concept of the disease based on our findings and an extensive review of the literature. We found that 79% of patients showed asymmetrical symptoms, 60% showed clinical or electrophysiological signs of lower motor neuron (LMN) involvement after a mean of 8.4 ± 5.0years, and extrapyramidal and/or non-motoric symptoms were frequently observed. Interestingly, none of the patients diagnosed with PLS fulfilled the diagnostic criteria proposed by Pringle etal. in 1992. Our data show that PLS as a disease entity is still not well enough defined and that there are different concepts about its clinical presentation. We believe that further prospective longitudinal studies are needed in order to refine diagnostic criteria to reflect current clinical practice. Furthermore, neuropathological and neuroimaging approaches might help to arrange PLS in the MND spectrum and its classification.

CHCHD10 mutations and motor neuron disease: the distribution in Finnish patients

Motor neuron disorders (MNDs) are a heterogeneous group of diseases that result from degeneration of motor neurons. If both upper and lower motor neurons (UMNs and LMNs) are affected, the...

Rare Variants in Neurodegeneration Associated Genes Revealed by Targeted Panel Sequencing in a German ALS Cohort.

Amyotrophic lateral sclerosis (ALS) is a progressive fatal multisystemic neurodegenerative disorder caused by preferential degeneration of upper and lower motor neurons. To further delineate the genetic architecture of the disease, we used comprehensive panel sequencing in a cohort of 80 German ALS patients. The panel covered 39 confirmed ALS genes and candidate genes, as well as 238 genes associated with other entities of the neurodegenerative disease spectrum. In addition, we performed repeat length analysis for C9orf72. Our aim was to (1) identify potentially disease-causing variants, to (2) assess a proposed model of polygenic inheritance in ALS and to (3) connect ALS with other neurodegenerative entities. We identified 79 rare potentially pathogenic variants in 27 ALS associated genes in familial and sporadic cases. Five patients had pathogenic C9orf72 repeat expansions, a further four patients harbored intermediate length repeat expansions. Our findings demonstrate that a genetic background of the disease can actually be found in a large proportion of seemingly sporadic cases and that it is not limited to putative most frequently affected genes such as C9orf72 or SOD1. Assessing the polygenic nature of ALS, we identified 15 patients carrying at least two rare potentially pathogenic variants in ALS associated genes including pathogenic or intermediate C9orf72 repeat expansions. Multiple variants might influence severity or duration of disease or could account for intrafamilial phenotypic variability or reduced penetrance. However, we could not observe a correlation with age of onset in this study. We further detected potentially pathogenic variants in other neurodegeneration associated genes in 12 patients, supporting the hypothesis of common pathways in neurodegenerative diseases and linking ALS to other entities of the neurodegenerative spectrum. Most interestingly we found variants in GBE1 and SPG7 which might represent differential diagnoses. Based on our findings, we recommend two-staged genetic testing for ALS in Germany in patients with familial and sporadic ALS, comprising C9orf72 repeat analysis followed by comprehensive panel sequencing including differential diagnoses that impair motor neuron function to meet the complexity of ALS genetics.

Matrin 3 variants are frequent in Italian ALS patients

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by loss of motor neurons in the primary motor cortex, brainstem, and spinal cord. Recently, missense variants in MATR3 were identified in familial and sporadic ALS patients, but very few additional ALS patients have been reported so far. The p.S85C MATR3 variant was previously associated to a different phenotype, namely a distal myopathy associated with dysphagia and dysphonia. Here, we assessed the contribution of MATR3 variants in a cohort of 322 Italian ALS patients. We identified 5 different missense MATR3 variants (p.Q66K, p.G153C, p.E664A, p.S707L, and p.N787S) in 6 patients (1.9%). None of our patients showed signs of myopathy at electrophysiological examination. Muscle biopsy, performed in 2 patients, showed neurogenic changes and normal nuclear staining with anti-matrin 3 antibody. Our results confirm that MATR3 variants are associated with ALS and suggest that they are more frequent in Italian ALS patients. Further studies are needed to elucidate the pathogenic significance of identified variants in sporadic and familial ALS.

Corticoefferent pathways in pure lower motor neuron disease: a diffusion tensor imaging study

Criteria for assessing upper motor neuron pathology in lower motor neuron disease (LMND) still remain major issues in clinical diagnosis. This study was designed to investigate patients with the clinical diagnosis of adult pure LMND by use of whole brain based diffusion tensor imaging (DTI) to delineate alterations of corticoefferent pathways in vivo. Comparison of fractional anisotropy (FA) maps was performed by whole brain-based spatial statistics for 37 LMND patients vs. 53 matched controls to detect white matter structural alterations. LMND patients were clinically differentiated in fast and slow progressors. Furthermore, tract specific alterations were investigated by fiber tracking techniques according to the staging hypothesis for amyotrophic lateral sclerosis (ALS). The analysis of white matter structural connectivity demonstrated widespread and characteristic patterns of alterations in patients with LMND, predominantly along the corticospinal tract (CST), with multiple clusters of regional FA reductions in the motor system at p<0.05 (corrected for multiple comparisons). Fast progressing LMND showed substantial CST involvement, while slow progressors showed less CST alterations. In the tract-specific analysis according to the ALS-staging pattern as suggested by Braak, fast progressing LMND showed significant alterations of ALS-related tract systems beyond the CST compared to slow progressors and controls. In clinically pure LMND patients, the involvement of corticoefferent fibers was demonstrated, in particular along the CST, supporting the hypothesis that LMND is a phenotypical variant of ALS. This finding suggests to treat these patients like ALS, including the opportunity to participate in clinical trials.

61. Drop Foot as initial symptom of pseudopolyneuritic variant motor neuron disease. A case report

Objective The pseudo-polyneuritic variant of amyotrophic lateral sclerosis can initially show a drop foot or distal weakness with achilles tendon arreflexia, simulating neuropathology. The early detection is very important as a part of motor neuron disease (MND) spectrum. Material and methods A 45 years old male, without other pathologies and clean clinical record history, presented progressive and ascendant left food weakness for the last 3 months, with subsequent weakness in contralateral foot and left hand one month ago. Also, he noted disseminated fasciculations. At neurological examination, we found normal strength in upper extremities, 4/5 proximal and 3/5 distal in lower extremities with 3/4 muscular stretching reflexes except bilateral achilles reflex (0/4). Presence of bilateral Tromner and fasciculations in all extremities were noted. Results Electromyography showed neuropathic pattern with disseminated active denervation in proximal and distal muscles in all extremities, and severe motor axonal degeneration in lower extremities, with median nerve asymmetry. Conclusions It is important to recognize this Motor Neuron disease variant because it might be confused with other clinical conditions such lumbar Plexopathy, Polyneuropathy, Peroneal Mononeuropathy, or distal Myopathy for the clinical presence of drop foot. Therefore, clinical record and Neurophysiological evaluation are very important for accurate diagnosis in this patient.

The clinical spectrum of sporadic and familial forms of frontotemporal dementia.

The term frontotemporal dementia (FTD) describes a clinically, genetically and pathologically diverse group of neurodegenerative disorders. Symptoms of FTD can present in individuals in their 20s through to their 90s, but the mean age at onset is in the sixth decade. The most common presentation is with a change in personality and impaired social conduct (behavioural variant FTD). Less frequently patients present with language problems (primary progressive aphasia). Both of these groups of patients can develop motor features consistent with either motor neuron disease (usually the amyotrophic lateral sclerosis variant) or parkinsonism (most commonly a progressive supranuclear palsy or corticobasal syndrome). In about a third of cases FTD is familial, with mutations in the progranulin, microtubule-associated protein tau and chromosome 9 open reading frame 72 genes being the major causes. Mutations in a number of other genes including TANK-binding kinase 1 are rare causes of familial FTD. This review aims to clarify the often confusing terminology of FTD, and outline the various clinical features and diagnostic criteria of sporadic and familial FTD syndromes. It will also discuss the current major challenges in FTD research and clinical practice, and potential areas for future research. This review clarifies the terminology of frontotemporal dementia (FTD) and summarizes the various clinical features and most recent diagnostic criteria of sporadic and familial FTD syndromes. It also discusses the current major challenges in FTD research and clinical practice, and highlights potential areas for future research.

Motor neuron disease with saccadic abnormalities similar to progressive supranuclear palsy

AbstractBackgroundIn recent years, a variety of clinical types of amyotrophic lateral sclerosis have come to be recognized. As some patients present with oculomotor abnormalities both clinically and pathologically, the progressive supranuclear palsy variant of amyotrophic lateral sclerosis has been proposed.AimTo describe atypical cases of motor neuron disease with abnormal extraocular movements mimicking progressive supranuclear palsy.MethodsWe present three motor neuron disease patients with slow saccades, who were aged 57, 63 and 62 years. Neurological examinations found vertical gaze palsy in two patients. The two patients who presented extrapyramidal signs were regarded as motor neuron disease with parkinsonism, whereas the other was diagnosed with amyotrophic lateral sclerosis. Their saccades were investigated by visually‐guided saccade and memory‐guided saccade tasks, and were compared with those of 14 age‐matched normal participants (60.3 ± 1.9 years).ResultsIn all these patients, the visually‐guided saccade latencies were significantly prolonged compared with normal participants, whereas the memory‐guided saccade latencies were not. The velocity and amplitude of saccades of the patients were significantly reduced in visually‐guided saccade and memory‐guided saccade in comparison with normal participants.ConclusionThe patterns of saccadic abnormalities in the patients were similar to those of progressive supranuclear palsy patients, suggesting that some patients with motor neuron disease show saccade abnormalities similar to those of progressive supranuclear palsy patients from the clinical and physiological perspective. Motor neuron disease with slow saccades and parkinsonism, as reported here, suggest the existence of progressive supranuclear palsy‐variant amyotrophic lateral sclerosis.

Gibbs Energy of Superoxide Dismutase Heterodimerization Accounts for Variable Survival in Amyotrophic Lateral Sclerosis.

The exchange of subunits between homodimeric mutant Cu, Zn superoxide dismutase (SOD1) and wild-type (WT) SOD1 is suspected to be a crucial step in the onset and progression of amyotrophic lateral sclerosis (ALS). The rate, mechanism, and ΔG of heterodimerization (ΔGHet) all remain undetermined, due to analytical challenges in measuring heterodimerization. This study used capillary zone electrophoresis to measure rates of heterodimerization and ΔGHet for seven ALS-variant apo-SOD1 proteins that are clinically diverse, producing mean survival times between 2 and 12 years (postdiagnosis). The ΔGHet of each ALS variant SOD1 correlated with patient survival time after diagnosis (R(2) = 0.98), with more favorable ΔGHet correlating with shorter survival by 4.8 years per kJ. Rates of heterodimerization did not correlate with survival time or age of disease onset. Metalation diminished the rate of subunit exchange by up to ∼38-fold but only altered ΔGHet by <1 kJ mol(-1). Medicinal targeting of heterodimer thermodynamics represents a plausible strategy for prolonging life in SOD1-linked ALS.

Absence of split hand in the flail arm variant of ALS

Flail arm syndrome (FAS), a variant of amyotrophic lateral sclerosis (ALS), has many similarities with upper limb onset ALS (UL-ALS). This study analyzed the compound muscle action potentials (CMAPs) recorded from abductor pollicis brevis (APB) and abductor digiti minimi (ADM) muscles in patients presenting these two types of ALS variants, compared to normal controls. The APB/ADM CMAP amplitude ratio was lower in the UL-ALS group (mean±SEM: 0.56±0.37), consistent with a split hand, compared to the FAS (1.05±0.29) or control (1.11±0.30) groups. An abnormally reduced APB/ADM CMAP amplitude ratio (<0.6) was found in 40% of UL-ALS patients and 11% of FAS patients. However, absent CMAP in the APB or extremely low APB/ADM CMAP amplitude ratio (<0.25) were observed only in UL-ALS patients (27%) and could be used as diagnostic criteria to differentiate UL-ALS from FAS variant.

110. Tongue’s motor evoked potentials in the diagnosis of Primary Lateral Sclerosis (PLS): Preliminary report

Primary Lateral Sclerosis (PLS) is an adult-onset neurodegenerative disorder due to a selective loss of precentral pyramidal neurons. Our purpose was to evaluate preferential impairment of pyramidal tract to bulbar muscles in patients with PLS and identify a valuable electrophysiological method to help clinicians in the differential diagnosis from Amyotrophic Lateral Sclerosis (ALS). We recorded Motor Evoked Potentials (MEPs) from tongue and anterior tibialis muscles in six patients with PLS and compared the results, in terms of Central Motor Conduction Time (CMCT), amplitude of MEPs and duration of controlateral silent period (cSP), with those obtained both from ten age-matched healthy volunteers and ten patients affected by ALS. For lower limbs, CMCT resulted significantly increased in PLS and ALS samples compared with healthy subjects (p < 0.01); we did not disclose any difference between ALS and PLS groups (p = 0.417). Instead for tongue’s recordings, CMCT, absolute amplitude of MEPs and cSP were significantly altered in PLS patients towards both ALS patients and healthy volunteers. Altogether tongue’s MEPs are selectively impaired in PLS. This technique could be helpful for the diagnosis of PLS and to differentiate patients with PLS from those affected by upper motor neuron-predominant variants of ALS.

Cortical contributions to the flail leg syndrome: Pathophysiological insights

Cortical hyperexcitability has been identified as an intrinsic feature of amyotrophic lateral sclerosis (ALS). Consequently, the aim of the present study was to determine whether cortical hyperexcitability formed the pathophysiological basis for the flail leg syndrome (FL), an atypical ALS variant. Cortical excitability studies were undertaken on 18 FL patients, using the threshold tracking transcranial magnetic stimulation (TMS) technique, and results were compared to healthy controls, upper and lower limb-onset ALS as well as bulbar-onset and the flail arm variant ALS. Results showed that cortical hyperexcitability was a feature of FL and was heralded by a significant reduction of short-interval intracortical inhibition (FL 7.2 ± 1.8%; controls 13.2 ± 0.8%, p <0.01) and cortical silent period (CSP) duration (FL 181.7 ± 10.8ms; controls 209.8 ± 3.4ms; p <0.05) along with an increase in motor evoked potential amplitude (FL 29.2 ± 5.1%; controls 18.9 ± 1.2%, p <0.05). The degree of cortical hyperexcitability was comparable between FL and other ALS phenotypes, defined by site of disease onset. In addition, the CSP duration correlated with biomarkers of peripheral neurodegeneration in FL. In conclusion, cortical hyperexcitability is a feature of the flail leg syndrome, being comparable to other ALS phenotypes. Importantly, cortical hyperexcitability correlates with neurodegeneration, and as such may contribute to the underlying pathophysiology in FL.

C9ORF72 GGGGCC Expanded Repeats Produce Splicing Dysregulation which Correlates with Disease Severity in Amyotrophic Lateral Sclerosis.

ObjectiveAn intronic GGGGCC-repeat expansion of C9ORF72 is the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The mechanism of neurodegeneration is unknown, but a direct effect on RNA processing mediated by RNA foci transcribed from the repeat sequence has been proposed.MethodsGene expression profiling utilised total RNA extracted from motor neurons and lymphoblastoid cell lines derived from human ALS patients, including those with an expansion of C9ORF72, and controls. In lymphoblastoid cell lines, expansion length and the frequency of sense and antisense RNA foci was also examined.ResultsGene level analysis revealed a number of differentially expressed networks and both cell types exhibited dysregulation of a network functionally enriched for genes encoding ‘RNA splicing’ proteins. There was a significant overlap of these genes with an independently generated list of GGGGCC-repeat protein binding partners. At the exon level, in lymphoblastoid cells derived from C9ORF72-ALS patients splicing consistency was lower than in lines derived from non-C9ORF72 ALS patients or controls; furthermore splicing consistency was lower in samples derived from patients with faster disease progression. Frequency of sense RNA foci showed a trend towards being higher in lymphoblastoid cells derived from patients with shorter survival, but there was no detectable correlation between disease severity and DNA expansion length.SignificanceUp-regulation of genes encoding predicted binding partners of the C9ORF72 expansion is consistent with an attempted compensation for sequestration of these proteins. A number of studies have analysed changes in the transcriptome caused by C9ORF72 expansion, but to date findings have been inconsistent. As a potential explanation we suggest that dynamic sequestration of RNA processing proteins by RNA foci might lead to a loss of splicing consistency; indeed in our samples measurement of splicing consistency correlates with disease severity.

Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy

GGGGCC repeat expansions of C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We and others have proposed that RNA transcribed from the repeat sequence is toxic via sequestration of RNA-binding factors. Both GGGGCC-repeat (sense) and CCCCGG-repeat (antisense) molecules are detectable by fluorescence in situ hybridisation as RNA foci, but their relative expression pattern within the CNS and contribution to disease has not been determined. Blinded examination of CNS biosamples from ALS patients with a repeat expansion of C9ORF72 showed that antisense foci are present at a significantly higher frequency in cerebellar Purkinje neurons and motor neurons, whereas sense foci are present at a significantly higher frequency in cerebellar granule neurons. Consistent with this, inclusions containing sense or antisense derived dipeptide repeat proteins were present at significantly higher frequency in cerebellar granule neurons or motor neurons, respectively. Immunohistochemistry and UV-crosslinking studies showed that sense and antisense RNA molecules share similar interactions with SRSF2, hnRNP K, hnRNP A1, ALYREF, and hnRNP H/F. Together these data suggest that, although sense and antisense RNA molecules might be expected to be equally toxic via their shared protein binding partners, distinct patterns of expression in various CNS neuronal populations could lead to relative differences in their contribution to the pathogenesis of neuronal injury. Moreover in motor neurons, which are the primary target of pathology in ALS, the presence of antisense foci (χ2, p < 0.00001) but not sense foci (χ2, p = 0.75) correlated with mislocalisation of TDP-43, which is the hallmark of ALS neurodegeneration. This has implications for translational approaches to C9ORF72 disease, and furthermore interacting RNA-processing factors and transcriptional activators responsible for antisense versus sense transcription might represent novel therapeutic targets.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1429-9) contains supplementary material, which is available to authorized users.

Exome sequencing of case-unaffected-parents trios reveals recessive and de novo genetic variants in sporadic ALS.

The contribution of genetic variants to sporadic amyotrophic lateral sclerosis (ALS) remains largely unknown. Either recessive or de novo variants could result in an apparently sporadic occurrence of ALS. In an attempt to find such variants we sequenced the exomes of 44 ALS-unaffected-parents trios. Rare and potentially damaging compound heterozygous variants were found in 27% of ALS patients, homozygous recessive variants in 14% and coding de novo variants in 27%. In 20% of patients more than one of the above variants was present. Genes with recessive variants were enriched in nucleotide binding capacity, ATPase activity, and the dynein heavy chain. Genes with de novo variants were enriched in transcription regulation and cell cycle processes. This trio study indicates that rare private recessive variants could be a mechanism underlying some case of sporadic ALS, and that de novo mutations are also likely to play a part in the disease.

Novel atomic force microscopy based biopanning for isolation of morphology specific reagents against TDP-43 variants in amyotrophic lateral sclerosis.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson's disease and beta-amyloid and tau in Alzheimer's disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson’s disease and beta-amyloid and tau in Alzheimer’s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.