Many neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and frontotemporal dementia, are proteinopathies that are associated with the aggregation and accumulation of misfolded proteins. While remarkable progress has been made in understanding the triggers of these conditions, several challenges have hampered the translation of preclinical therapies targeting pathways downstream of the initiating proteinopathies. Clinical trials in symptomatic patients using therapies directed toward initiating trigger events have met with little success, prompting concerns that such therapeutics may be of limited efficacy when used in advanced stages of the disease rather than as prophylactics. Herein, we discuss gaps in our understanding of the pathological processes downstream of the trigger and potential strategies to identify common features of the downstream degenerative cascade in multiple CNS proteinopathies, which could potentially lead to the development of common therapeutic targets for multiple disorders.
By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures.
In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures.
Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells.
Palmitoyl acyl transferases (PATs) play a critical role in protein trafficking and function. Huntingtin interacting protein 14 (HIP14) is a PAT that acts on proteins associated with neuronal transmission, suggesting that deficient protein palmitoylation by HIP14, which occurs in the YAC128 model of Huntington’s disease (HD), might have deleterious effects on neurobehavioral processing. HIP14 knockout mice show biochemical and neuropathological changes in the striatum, a forebrain region affected by HD that guides behavioral choice and motor flexibility. Thus, we evaluated the performance of these mice in two tests of motor ability: nest-building and plus maze turning behavior. Relative to wild-type controls, HIP14 knockout mice show impaired nest building and decreased turning in the plus maze. When we recorded the activity of striatal neurons during plus-maze performance, we found faster firing rates and dysregulated spike bursting in HIP14 knockouts compared to wild-type. There was also less correlated firing between simultaneously recorded neuronal pairs in the HIP14 knockouts. Overall, our results indicate that HIP14 is critically involved in behavioral modulation of striatal processing. In the absence of HIP14, striatal neurons become dysfunctional, leading to impaired motor behavior.
Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin protein. Neuropathology in the basal ganglia and in the cerebral cortex has been linked to the motor and cognitive symptoms whereas recent work has suggested that the hypothalamus might be involved in the metabolic dysfunction. Several mouse models of HD that display metabolic dysfunction have hypothalamic pathology, and expression of mutant huntingtin in the hypothalamus has been causally linked to the development of metabolic dysfunction in mice. Although the pathogenic mechanisms by which mutant huntingtin exerts its toxic functions in the HD brain are not fully known, several studies have implicated a role for the lysososomal degradation pathway of autophagy. Interestingly, changes in autophagy in the hypothalamus have been associated with the development of metabolic dysfunction in wild-type mice. We hypothesized that expression of mutant huntingtin might lead to changes in the autophagy pathway in the hypothalamus in mice with metabolic dysfunction. We therefore investigated whether there were changes in basal levels of autophagy in a mouse model expressing a fragment of 853 amino acids of mutant huntingtin selectively in the hypothalamus using a recombinant adeno-associate viral vector approach as well as in the transgenic BACHD mice. We performed qRT-PCR and Western blot to investigate the mRNA and protein expression levels of selected autophagy markers. Our results show that basal levels of autophagy are maintained in the hypothalamus despite the presence of metabolic dysfunction in both mouse models. Furthermore, although there were no major changes in autophagy in the striatum and cortex of BACHD mice, we detected modest, but significant differences in levels of some markers in mice at 12 months of age. Taken together, our results indicate that overexpression of mutant huntingtin in mice do not significantly perturb basal levels of autophagy.
Mutations in the gene encoding superoxide dismutase 1 (SOD1) account for about 20% of the cases of familial amyotrophic lateral sclerosis (fALS). It is well established that mutations in SOD1, associated with fALS, heighten the propensity of the protein to misfold and aggregate. Although aggregation appears to be a factor in the toxicity of mutant SOD1s, the precise nature of this toxicity has not been elucidated. A number of other studies have now firmly established that raising the levels of wild-type (WT) human SOD1 (hSOD1) proteins can in some manner augment the toxicity of mutant hSOD1 proteins. However, a recent study demonstrated that raising the levels of WT-hSOD1 did not affect disease in mice that harbor a mouse Sod1 gene (mSod1) encoding a well characterized fALS mutation (G86R). In the present study, we sought a potential explanation for the differing effects with WT-hSOD1 on the toxicity of mutant hSOD1 versus mutant mSod1. In the cell culture models used here, we observe poor interactions between WT-hSOD1 and misfolded G86R-mSod1, possibly explaining why over-expression of WT-hSOD1 does not synergize with mutant mSod1 to accelerate the course of the disease in mice.
Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r2=.67), motor neuron counts (attrition) (p<.0001, r2=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.
Transgenic mice that express mutant amyloid precursor protein (APPsi) using tet-Off vector systems provide an alternative model for assessing short- and long-term effects of Aβ-targeting therapies on phenotypes related to the deposition of Alzheimer-type amyloid. Here we use such a model, termed APPsi:tTA, to determine what phenotypes persist in mice with high amyloid burden after new production of APP/Aβ has been suppressed. We find that 12-13 month old APPsi:tTA mice are impaired in cognitive tasks that assess short- and long-term memories. Acutely suppressing new APPsi/Aβ production produced highly significant improvements in performance short-term spatial memory tasks; which upon continued suppression translated to superior performance in more demanding tasks that assess long-term spatial memory and working memory. Deficits in episodic-like memory and cognitive flexibility, however, were more persistent. Arresting mutant APPsi production caused a rapid decline in the brain levels of soluble APP ectodomains, full-length APP, and APP C-terminal fragments. As expected, amyloid deposits persisted after new APP/Aβ production was inhibited whereas, unexpectedly, we detected persistent pools of solubilizable, relatively mobile, Aβ42. Additionally, we observed persistent levels of Aβ immunoreactive entities that were of a size consistent with SDS-resistant oligomeric assemblies. Thus, in this model with significant amyloid pathology, a rapid amelioration of cognitive deficits was observed despite persistent levels of oligomeric Aβ assemblies and low, but detectable solubilizable Aβ42 peptides. These findings implicate complex relationships between accumulating Aβ and activities of APP, soluble APP ectodomains, and/or APP CTFs in mediating cognitive deficits in this model of amyloidosis.
Mutations in SOD1 cause FALS. The Cu binding capacity of SOD1 has spawned hypotheses that implicate metal-mediated production of reactive species as a potential mechanism of toxicity. In past experiments, we have tested such hypotheses by mutating residues in SOD1 that normally coordinate the binding of Cu, finding that such mutants retain the capacity to induce motor neuron disease. We now describe the lack of disease in mice that express a variant of human SOD1 in which residues that coordinate the binding of Cu and Zn have been mutated (SODMD). SODMD encodes 3 disease-causing and 4 experimental mutations that ultimately eliminate all histidines involved in the binding of metals; and includes one disease-causing and one experimental mutation that eliminate secondary metal binding at C6 and C111. We show that the combined effect of these mutations produces a protein that is unstable but does not aggregate on its own, is not toxic, and does not induce disease when co-expressed with high levels of wild-type SOD1. In cell culture models, we determine that the combined mutation of C6 and C111 to G and S, respectively, dramatically reduces the aggregation propensity of SODMD and may account for the lack of toxicity for this mutant.
superoxide dismutase 1; motor neuron disease; transgenic mouse models; protein aggregation
Recent research in Alzheimer’s disease (AD) field has been focused on the potential role of the amyloid-β protein that is derived from the transmembrane amyloid precursor protein (APP) in directly mediating cognitive impairment in AD. Transgenic mouse models overexpressing APP develop robust AD-like amyloid pathology in the brain and show various levels of cognitive decline. In the present study, we examined the cognition of the BRI2-Aβ transgenic mouse model in which secreted extracellular Aβ1-40, Aβ1-42 or both Aβ1-40/Aβ1-42 peptides are generated from the BRI-Aβ fusion proteins encoded by the transgenes. BRI2-Aβ mice produce high levels of Aβ peptides and BRI2-Aβ1-42 mice develop amyloid pathology that is similar to the pathology observed in mutant human APP transgenic models.
Using established behavioral tests that reveal deficits in APP transgenic models, BRI2-Aβ1-42 mice showed completely intact cognitive performance at ages both pre and post amyloid plaque formation. BRI2-Aβ mice producing Aβ1-40 or both peptides were also cognitively intact.
These data indicate that high levels of Aβ1-40 or Aβ1-42, or both produced in the absence of APP overexpression do not reproduce memory deficits observed in APP transgenic mouse models. This outcome is supportive of recent data suggesting that APP processing derivatives or the overexpression of full length APP may contribute to cognitive decline in APP transgenic mouse models. Alternatively, Aβ aggregates may impact cognition by a mechanism that is not fully recapitulated in these BRI2-Aβ mouse models.
Alzheimer’s disease; Mouse models; Amyloid-β; Amyloid plaques; Cognition
Frontotemporal lobar degeneration (FTLD) has been subdivided based on the main pathology found in the brains of affected individuals. When the primary pathology is aggregated, hyperphosphorylated tau, the pathological diagnosis is FTLD-tau. When the primary pathology is cytoplasmic and/or nuclear aggregates of phosphorylated TAR-DNA-binding protein (TDP-43), the pathological diagnosis is FTLD-TDP. Notably, TDP-43 pathology can also occur in conjunction with a number of neurodegenerative disorders; however, unknown environmental and genetic factors may regulate this TDP-43 pathology. Using transgenic mouse models of several diseases of the central nervous system, we explored whether a primary proteinopathy might secondarily drive TDP-43 proteinopathy. We found abnormal, cytoplasmic accumulation of phosphorylated TDP-43 specifically in two tau transgenic models, but TDP-43 pathology was absent in mouse models of Aβ deposition, α-synucleinopathy or Huntington’s disease. Though tau pathology showed considerable overlap with cytoplasmic, phosphorylated TDP-43, tau pathology generally preceded TDP-43 pathology. Biochemical analysis confirmed the presence of TDP-43 abnormalities in the tau mice, which showed increased levels of high molecular weight, soluble TDP-43 and insoluble full-length and ~35 kD TDP-43. These data demonstrate that the neurodegenerative cascade associated with a primary tauopathy in tau transgenic mice can also promote TDP-43 abnormalities. These findings provide the first in vivo models to understand how TDP-43 pathology may arise as a secondary consequence of a primary proteinopathy.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-013-1123-8) contains supplementary material, which is available to authorized users.
Tau; TDP-43; Mouse; Transgenic; Neuropathology, tauopathy; TDP-43 proteinopathies
Peripheral blood Apolipoprotein E (ApoE) levels have been proposed as biomarkers of Alzheimer’s disease (AD), but previous studies on levels of ApoE in blood remain inconsistent. This meta-analysis was designed to re-examine the potential role of peripheral ApoE in AD diagnosis and its potential value as a candidate biomarker.
We conducted a systematic literature search of MEDLINE, EMBASE, the Cochrane library, and BIOSIS previews for case-control studies measuring ApoE levels in serum or plasma from AD subjects and healthy controls. The pooled weighted mean difference (WMD) and 95% confidence interval (CI) were used to estimate the association between ApoE levels and AD risk.
Eight studies with a total of 2250 controls and 1498 AD cases were identified and analyzed. The pooled WMD from a random-effect model of AD participants compared with the healthy controls was −5.59 mg/l (95% CI: [−8.12, −3.06]). The overall pattern in WMD was not varied by characteristics of study, including age, country, assay method, publication year, and sample type.
Our meta-analysis supports a lowered level of blood ApoE in AD patients, and indicates its potential value as an important risk factor for AD. Further investigation employing standardized assay for ApoE measurement are still warranted to uncover the precise role of ApoE in the pathophysiology of AD.
Anti-GD2 antibody is a proven therapy for GD2-postive neuroblastoma. Monoclonal antibodies against GD2, such as chimeric mAb ch14.18, have become benchmarks for neuroblastoma therapies. Pain, however, can limit immunotherapy with anti-GD2 therapeutic antibodies like ch14.18. This adverse effect is attributed to acute inflammation via complement activation on GD2-expressing nerves. Thus, new strategies are needed for the development of treatment intensification strategies to improve the outcome of these patients.
We established the mouse-human chimeric antibody c.8B6 specific to OAcGD2 in order to reduce potential immunogenicity in patients and to fill the need for a selective agent that can kill neuroblastoma cells without inducing adverse neurological side effects caused by anti-GD2 antibody immunotherapy. We further analyzed some of its functional properties compared with anti-GD2 ch14.18 therapeutic antibody. With the exception of allodynic activity, we found that antibody c.8B6 shares the same anti-neuroblastoma attributes as therapeutic ch14.18 anti-GD2 mAb when tested in cell-based assay and in vivo in an animal model.
The absence of OAcGD2 expression on nerve fibers and the lack of allodynic properties of c.8B6–which are believed to play a major role in mediating anti-GD2 mAb dose-limiting side effects–provide an important rationale for the clinical application of c.8B6 in patients with high-risk neuroblastoma.
Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites.
Cognitive impairment is associated with a negative prognosis in amyotrophic lateral sclerosis (ALS), as well as with clinical specificity. We investigate neuropsychological function in ALS patients without known genetic mutations in a Korean tertiary clinic.
Three hundred and eighteen patients were enrolled in a prospective longitudinal cohort from September 2008 to February 2012. At the time of diagnosis of sporadic ALS, we carried out genetic and comprehensive neuropsychological tests on all patients, and collected demographic and clinical characteristics. Six cognitive domains, namely executive function, attention, language, calculation, visuospatial function and memory were evaluated. ANOVA and t-tests were used to assess differences in clinical characteristics and neuropsychological parameters between sporadic ALS patients. The Kaplan-Meier method and Cox proportional hazard model were used for survival analysis.
One hundred and sixty-six patients were categorized into five subtypes: normal cognition (ALS pure), cognitive impairment (ALSci), behavioral impairment (ALSbi), frontotemporal dementia (ALS-FTD), and other types of dementia. Seventy patients (70/166, 42.2%) were cognitively or behaviorally impaired. Among the impaired patients, eight (8/166, 4.8%) had FTD-type dementia and one (1/166, 0.6%) was Alzheimer's disease-type. The ALS patients with cognitive impairment (ALSci) and with FTD (ALS-FTD) were more severely impaired in executive function, attention, language and memory than the cognitively intact ALS patients (ALS pure). In a survival analysis, ALSci (β = 1.925, p = 0.025) and ALS-FTD groups (β = 4.150, p = 0.019) tended to have shorter survival than the ALS pure group.
About half of ALS patients without known genetic variation have cognitive or behavioral impairment. ALS patients with cognitive abnormalities, especially FTD, have a poorer prognosis than those without cognitive impairment. In neuropsychological profiling, executive tasks were effective in identifying cognitive impairment in the ALS patients. It would be useful for clinicians to classify ALS according to neuropsychological profiles, and screen for subtle cognitive impairment.
The synucleins are a family of natively unstructured proteins consisting of α-, β-, and γ-synuclein which are primarily expressed in neurons. They have been linked to a wide variety of pathologies, including neurological disorders, such as Parkinson’s disease (α-synuclein) and dementia with Lewy bodies (α- and β-synuclein), as well as various types of cancers (γ-synuclein). Self-association is a key pathological feature of many of these disorders, with α-synuclein having the highest propensity to form aggregates, while β-synuclein is the least prone. Here, we used a combination of fluorescence correlation spectroscopy and single molecule Förster resonance energy transfer to compare the intrinsic dynamics of different regions of all three synuclein proteins to investigate any correlation with putative functional or dysfunctional interactions. Despite a relatively high degree of sequence homology, we find that individual regions sample a broad range of diffusion coefficients, differing by almost a factor of four. At low pH, a condition that accelerates aggregation of α-synuclein, on average smaller diffusion coefficients are measured, supporting a hypothesis that slower intrachain dynamics may be correlated with self-association. Moreover, there is a surprising inverse correlation between dynamics and bulkiness of the segments. Aside from this observation, we could not discern any clear relationship between the physico-chemical properties of the constructs and their intrinsic dynamics. This work suggests that while protein dynamics may play a role in modulating self-association or interactions with other binding partners, other factors, particularly the local cellular environment, may be more important.
The split-hand phenomenon, a specific feature of amyotrophic lateral sclerosis (ALS), refers to preferential wasting of abductor pollicis brevis (APB) and first dorsal interosseous (FDI) with relative preservation of abductor digiti minimi (ADM). The pathophysiological mechanisms underlying the split-hand phenomenon remain elusive and resolution of this issue would provide unique insights into ALS pathophysiology. Consequently, the present study dissected out the relative contribution of cortical and peripheral processes in development of the split-hand phenomenon in ALS. Cortical and axonal excitability studies were undertaken on 26 ALS patients, with motor responses recorded over the APB, FDI and ADM muscles. Results were compared to 21 controls. Short interval intracortical inhibition (SICI), a biomarker of cortical excitability, was significantly reduced across the range of intrinsic hand muscles (APBSICI ALS 0.3±2.0%, APBSICI controls 16.0±1.9%, P<0.0001; FDISICI ALS 2.7±1.7%, FDI SICI controls 14.8±1.9%, P<0.0001; ADMSICI ALS 2.6±1.5%, ADM SICI controls 9.7±2.2%, P<0.001), although the reduction was most prominent when recorded over APB/FDI. Changes in SICI were accompanied by a significant increase in motor evoked potential amplitude and reduction of cortical silent period duration, all indicative of cortical hyperexcitability, and these were most prominent from the APB/FDI. At a peripheral level, a significant increase in strength-duration time constant and reduction in depolarising threshold electrotonus were evident in ALS, although these changes did not follow a split-hand distribution. Cortical dysfunction contributed to development of the split-hand in ALS, thereby implying an importance of cortical hyperexcitability in ALS pathogenesis.
Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.
Pathologic aggregates of superoxide dismutase 1 (SOD1) harboring mutations linked to familial amyotrophic lateral sclerosis (fALS) have been shown to contain aberrant intermolecular disulfide cross-links. In prior studies, we observed that intermolecular bonding was not necessary in the formation of detergent- insoluble SOD1 complexes by mutant SOD1, but we were unable to assess whether this type of bonding may be important for pathologic inclusion formation. In the present study, we visually assess the formation of large inclusions by fusing mutant SOD1 to yellow fluorescent protein (YFP).
Experimental constructs possessing mutations at all cysteine residues in SOD1 (sites 6, 57, 111, and 146 to F,S,Y,R or G,S,Y,R, respectively) were shown to maintain a high propensity of inclusion formation despite the inability to form disulfide cross-links. Interestingly, although aggregates form when all cysteines were mutated, double mutants of the ALS mutation C6G with an experimental mutation C111S exhibited low aggregation propensity.
Overall, this study is an extension of previous work demonstrating that cysteine residues in mutant SOD1 play a role in modulating aggregation and that intermolecular disulfide bonds are not required to produce large intracellular inclusion-like structures.
Presenilin 1 (PSEN1) encodes the catalytic subunit of γ-secretase, and PSEN1 mutations are the most common cause of early onset familial Alzheimer's disease (FAD). In order to elucidate pathways downstream of PSEN1, we characterized neural progenitor cells (NPCs) derived from FAD mutant PSEN1 subjects. Thus, we generated induced pluripotent stem cells (iPSCs) from affected and unaffected individuals from two families carrying PSEN1 mutations. PSEN1 mutant fibroblasts, and NPCs produced greater ratios of Aβ42 to Aβ40 relative to their control counterparts, with the elevated ratio even more apparent in PSEN1 NPCs than in fibroblasts. Molecular profiling identified 14 genes differentially-regulated in PSEN1 NPCs relative to control NPCs. Five of these targets showed differential expression in late onset AD/Intermediate AD pathology brains. Therefore, in our PSEN1 iPSC model, we have reconstituted an essential feature in the molecular pathogenesis of FAD, increased generation of Aβ42/40, and have characterized novel expression changes.
Recent studies have implicated an N-terminal caspase-6 cleavage product of mutant huntingtin (htt) as an important mediator of toxicity in Huntington's disease (HD). To directly assess the consequences of such fragments on neurologic function, we produced transgenic mice that express a caspase-6 length N-terminal fragment of mutant htt (N586) with both normal (23Q) and disease (82Q) length glutamine repeats. In contrast to mice expressing N586-23Q, mice expressing N586-82Q accumulate large cytoplasmic inclusion bodies that can be visualized with antibodies to epitopes throughout the N586 protein. However, biochemical analyses of aggregated mutant huntingtin in these mice demonstrated that the inclusion bodies are composed largely of a much smaller htt fragment (terminating before residue 115), with lesser amounts of full-length N586-82Q fragments. Mice expressing the N586-82Q fragment show symptoms typical of previously generated mice expressing mutant huntingtin fragments, including failure to maintain weight, small brain weight and reductions in specific mRNAs in the striatum. Uniquely, these N586-82Q mice develop a progressive movement disorder that includes dramatic deficits in motor performance on the rotarod and ataxia. Our findings suggest that caspase-6-derived fragments of mutant htt are capable of inducing novel HD-related phenotypes, but these fragments are not terminal cleavage products as they are subject to further proteolysis. In this scenario, mutant htt fragments derived from caspase 6, or possibly other proteases, could mediate HD pathogenesis via a ‘hit and run' type of mechanism in which caspase-6, or other larger N-terminal fragments, mediate a neurotoxic process before being cleaved to a smaller fragment that accumulates pathologically.
Mutations in superoxide dismutase 1 (SOD1, EC 220.127.116.11) cause familial amyotrophic lateral sclerosis (fALS); with aggregated forms of mutant protein accumulating in spinal cord tissues of transgenic mouse models and human patients. Mice over-expressing wild-type human SOD1 (WT hSOD1) do not develop ALS-like disease, but co-expression of WT enzyme at high levels with mutant SOD1 accelerates the onset of motor neuron disease compared to mice expressing mutant hSOD1 alone. Spinal cords of mice expressing both proteins contain aggregated forms of mutant protein and, in some cases, evidence of co-aggregation of WT hSOD1 enzyme. In the present study, we used a cell culture model of mutant SOD1 aggregation to examine how the presence of WT SOD1 affects mutant protein aggregation, finding that co-expression of WT SOD1, human (hSOD1) or mouse (mSOD1), delayed the formation of mutant hSOD1 aggregates; in essence appearing to slow the aggregation rate. In some combinations of WT and mutant hSOD1 co-expression, the aggregates that did eventually form appeared to contain WT hSOD1 protein. However, WT mSOD1 did not co-aggregate with mutant hSOD1 despite displaying a similar ability to slow mutant hSOD1 aggregation. Together, these studies indicate that WT SOD1 (human or mouse), when expressed at levels equivalent to the mutant protein, modulates aggregation of FALS-mutant hSOD1.
superoxide; dismutase; aggregation; amyotrophic lateral sclerosis
Huntington’s disease (HD) is caused by the expansion of a CAG repeat in the huntingtin (HTT) gene. The R6 mouse models of HD express a mutant version of exon 1 HTT and typically develop motor and cognitive impairments, a widespread huntingtin (HTT) aggregate pathology and brain atrophy. Unlike the more commonly used R6/2 mouse line, R6/1 mice have fewer CAG repeats and, subsequently, a less rapid pathological decline. Compared to the R6/2 line, fewer descriptions of the progressive pathologies exhibited by R6/1 mice exist. The association between the molecular and cellular neuropathology with brain atrophy, and with the development of behavioral phenotypes remains poorly understood in many models of HD. In attempt to link these factors in the R6/1 mouse line, we have performed detailed assessments of behavior and of regional brain abnormalities determined through longitudinal, in vivo magnetic resonance imaging (MRI), as well as an end-stage, ex vivo MRI study and histological assessment. We found progressive decline in both motor and non-motor related behavioral tasks in R6/1 mice, first evident at 11 weeks of age. Regional brain volumes were generally unaffected at 9 weeks, but by 17 weeks there was significant grey matter atrophy. This age-related brain volume loss was validated using a more precise, semi-automated Tensor Based morphometry assessment. As well as these clear progressive phenotypes, mutant HTT (mHTT) protein, the hallmark of HD molecular pathology, was widely distributed throughout the R6/1 brain and was accompanied by neuronal loss. Despite these seemingly concomitant, robust pathological phenotypes, there appeared to be little correlation between the three main outcome measures: behavioral performance, MRI-detected brain atrophy and histopathology. In conclusion, R6/1 mice exhibit many features of HD, but the underlying mechanisms driving these clear behavioral disturbances and the brain volume loss, still remain unclear.
The molecular mechanisms by which polyglutamine (polyQ)-expanded huntingtin (Htt) causes neurodegeneration in Huntington's disease (HD) remain unclear. The malfunction of cellular proteostasis has been suggested as central in HD pathogenesis and also as a target of therapeutic interventions for the treatment of HD. We present results that offer a previously unexplored perspective regarding impaired proteostasis in HD. We find that, under non-stress conditions, the proteostatic capacity of cells expressing full length polyQ-expanded Htt is adequate. Yet, under stress conditions, the presence of polyQ-expanded Htt impairs the heat shock response, a key component of cellular proteostasis. This impaired heat shock response results in a reduced capacity to withstand the damage caused by cellular stress. We demonstrate that in cells expressing polyQ-expanded Htt the levels of heat shock transcription factor 1 (HSF1) are reduced, and, as a consequence, these cells have an impaired a heat shock response. Also, we found reduced HSF1 and HSP70 levels in the striata of HD knock-in mice when compared to wild-type mice. Our results suggests that full length, non-aggregated polyQ-expanded Htt blocks the effective induction of the heat shock response under stress conditions and may thus trigger the accumulation of cellular damage during the course of HD pathogenesis.
Cerebellar Purkinje neurons (PNs) possess a well characterized propensity to fuse with bone marrow-derived cells (BMDCs), producing heterokaryons with Purkinje cell identities. This offers the potential to rescue/repair at risk or degenerating PNs in the inherited ataxias, including Spinocerebellar Ataxia 1 (SCA1), by introducing therapeutic factors through BMDCs to potentially halt or reverse disease progression. In this study, we combined gene therapy and a stem cell-based treatment to attempt repair of at-risk PNs through cell-cell fusion in a Sca1154Q/2Q knock-in mouse model. BMDCs enriched for the hematopoietic stem cell (HSC) population were genetically modified using adeno-associated viral vector 7 (AAV7) to carry SCA1 modifier genes and transplanted into irradiated Sca1154Q/2Q mice. Binucleated Purkinje heterokaryons with sex-mismatched donor Y chromosomes were detected and successfully expressed the modifier genes in vivo. Potential effects of the new genome within Purkinje heterokaryons were evaluated using nuclear inclusions (NIs) as a biological marker to reflect possible modifications of the SCA1 disease process. An overall decrease in number of NIs and an increase in the number of surviving PNs were observed in treated Sca1154Q/2Q. Furthermore, Bergmann glia were found to have fusogenic potential with the donor population and reveal another potential route of therapeutic entry into at-risk cells of the SCA1 cerebellum. This study presents a first step towards a proof of principle that combines somatic cellular fusion events with a neuroprotective gene therapy approach for providing potential neuronal protection/repair in a variety of neurodegenerative disorders.
Spinocerebellar Ataxia 1; Bone marrow derived cells; Hematopoietic stem cells; Gene therapy; AAV; Stem cell fusion