Clinico-Pathological Correlation in Progressive Ataxia and Palatal Tremor: A Novel Tauopathy.

Palatal tremor (PT) is an uncommon movement disorder that has been subdivided into essential and symptomatic forms. A distinct subgroup of the symptomatic form presents with progressive ataxia and PT. The histopathology of progressive ataxia and PT has not been previously determined. This study consisted of a clinical review, histopathology, and electron microscopy of the brain of a man with progressive ataxia and PT. The inferior olivary hypertrophy was symmetrical and homogenous, and no focal pathologic lesions could be identified in the brainstem. Insoluble tau deposits were found in neurons, exclusively infratentorially. We present the clinical and pathological evaluation of a case of progressive ataxia and PT that provide evidence for a unique form of 4R tauopathy.

Inhibition of protein deubiquitination by PR-619 activates the autophagic pathway in OLN-t40 oligodendroglial cells.

Protein aggregate formation may be the result of an impairment of the protein quality control system, e.g., the ubiquitin proteasome system (UPS) and the lysosomal autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin and deubiquitinated before the substrates are proteolytically degraded. Deubiquitination is performed by a large family of proteases, the deubiquitinating enzymes (DUBs). DUBs display a variety of functions and their inhibition may have pathological consequences. Using the broad specificity DUB inhibitor PR-619 we previously have shown that DUB inhibition leads to an overload of ubiquitinated proteins, to protein aggregate formation and subsequent inhibition of the UPS. This study was undertaken to investigate whether PR-619 modulates autophagic functions to possibly compensate the failure of the proteasomal system. Using the oligodendroglial cell line OLN-t40 and a new oligodendroglial cell line stably expressing GFP-LC3, we show that DUB inhibition leads to the activation of autophagy and to the recruitment of LC3 and of the ubiquitin binding protein p62 to the forming aggresomes without impairing the autophagic flux. Furthermore, PR-619 induced the transport of lysosomes to the forming aggregates in a process requiring an intact microtubule network. Further stimulation of autophagy by rapamycin did not prevent PR-619 aggregate formation but rather exerted cytotoxic effects. Hence, inhibition of DUBs by PR-619 activated the autophagic pathway supporting the hypothesis that the UPS and the autophagy-lysosomal pathway are closely linked together.

Exogenous BDNF enhances the integration of chronically injured axons that regenerate through a peripheral nerve grafted into a chondroitinase-treated spinal cord injury site.

Although axons lose some of their intrinsic capacity for growth after their developmental period, some axons retain the potential for regrowth after injury. When provided with a growth-promoting substrate such as a peripheral nerve graft (PNG), severed axons regenerate into and through the graft; however, they stop when they reach the glial scar at the distal graft-host interface that is rich with inhibitory chondroitin sulfate proteoglycans. We previously showed that treatment of a spinal cord injury site with chondroitinase (ChABC) allows axons within the graft to traverse the scar and reinnervate spinal cord, where they form functional synapses. While this improvement in outgrowth was significant, it still represented only a small percentage (<20%) of axons compared to the total number of axons that regenerated into the PNG. Here we tested whether providing exogenous brain-derived neurotrophic factor (BDNF) via lentivirus in tissue distal to the PNG would augment regeneration beyond a ChABC-treated glial interface. We found that ChABC treatment alone promoted axonal regeneration but combining ChABC with BDNF-lentivirus did not increase the number of axons that regenerated back into spinal cord. Combining BDNF with ChABC did increase the number of spinal cord neurons that were trans-synaptically activated during electrical stimulation of the graft, as indicated by c-Fos expression, suggesting that BDNF overexpression improved the functional significance of axons that did reinnervate distal spinal cord tissue.

Polyvinyl alcohol-polyvinyl pyrrolidone thin films provide local short-term release of anti-inflammatory agents post spinal cord injury.

Spinal cord injury (SCI) triggers a large inflammatory response that results in exacerbated tissue damage. Locally delivering anti-inflammatory drugs could mitigate this secondary wave of degeneration. The mitogen-activated protein kinase family members p38 and c-Jun N-terminal kinase (JNK) play important roles in the inflammatory response and cell death. We propose that the use of polymer thin films, made of polyvinyl alcohol and polyvinyl pyrrolidone blends (PVA-PVP), can be used to provide local release of inhibitors to p38 and JNK post-SCI. Release studies performed in vitro confirmed the inhibitors could be released from the film for up to 7 days. The thin film was also tested for its surgical feasibility using a cervical contusion model of SCI in adult female rats. Films with or without the inhibitors were placed subdurally over the injury site immediately following SCI. Animals were sacrificed 5 days post-SCI and spinal cord tissue above and below the injury site was harvested. Additionally, films were removed for analysis. Scanning electron microscopy confirmed the anti-fouling properties of the PVA-PVP film. Tissue histology confirmed that the films themselves did not generate a large immune response, but they did compress the tissue slightly at its placement above the injury site. Finally, quantitative Western blot analysis determined the films loaded with p38 and JNK inhibitors delivered bioactive agents to the injury site and resulted in a significantly decreased amount of pro-cell death proteins. These data indicate that PVA-PVP films can be used to effectively deliver drugs to a SCI site.

The small molecule inhibitor PR-619 of deubiquitinating enzymes affects the microtubule network and causes protein aggregate formation in neural cells: implications for neurodegenerative diseases.

A pathological hallmark of many neurodegenerative diseases is the aggregation of proteins. Protein aggregate formation may be linked to a failure of the ubiquitin proteasome system (UPS) and/or the autophagy pathway. The UPS involves the ubiquitination of proteins followed by proteasomal degradation. Deubiquitination of target proteins is performed by proteases called deubiquitinating proteins (DUBs). Inhibition of DUBs may lead to the dysregulation of homeostasis and have pathological consequences. To assess the effects of DUB-inhibition, we have used the oligodendroglial cell line, OLN-t40, stably expressing the longest human tau isoform. Cells were incubated with PR-619, a broad-range, reversible inhibitor of ubiquitin isopeptidases. Incubation with PR-619 led to morphological changes, the upregulation of heat shock proteins (HSP), including HSP70 and αB-crystallin, and to protein aggregates near the MTOC, containing ubiquitin, HSPs, and the ubiquitin binding protein p62, which may provide a link between the UPS and autophagy. Thus, inhibition of DUB activity caused stress responses and the formation of protein aggregates resembling pathological inclusions observed in aggregopathies. Furthermore, PR-619 led to the stabilization of the microtubule network, possibly through the modulation of tau phosphorylation, and small tau deposits assembled near the MTOC. Hence, organization and integrity of the cytoskeleton were affected, which is particularly important for the maintenance of the cellular architecture and intracellular transport processes, and essential for the functionality and survival of neural cells. Our data demonstrate that DUB inhibitors provide a useful tool to elucidate the manifold mechanisms of DUB functions in cells and their dysregulation in neurodegenerative diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Acute and prolonged hindlimb exercise elicits different gene expression in motoneurons than sensory neurons after spinal cord injury.

We examined gene expression in the lumbar spinal cord and the specific response of motoneurons, intermediate gray and proprioceptive sensory neurons after spinal cord injury and exercise of hindlimbs to identify potential molecular processes involved in activity dependent plasticity. Adult female rats received a low thoracic transection and passive cycling exercise for 1 or 4weeks. Gene expression analysis focused on the neurotrophic factors: brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and their receptors because of their potential roles in neural plasticity. We also examined expression of genes involved in the cellular response to injury: heat shock proteins (HSP) -27 and -70, glial fibrillary acidic protein (GFAP) and caspases -3, -7, and -9. In lumbar cord samples, injury increased the expression of mRNA for TrkB, all three caspases and the HSPs. Acute and prolonged exercise increased expression of mRNA for the neurotrophic factors BDNF and GDNF, but not their receptors. It also increased HSP expression and decreased caspase-7 expression, with changes in protein levels complimentary to these changes in mRNA expression. Motoneurons and intermediate gray displayed little change in mRNA expression following injury, but acute and prolonged exercise increased levels of mRNA for BDNF, GDNF and NT-4. In large DRG neurons, mRNA for neurotrophic factors and their receptors were largely unaffected by either injury or exercise. However, caspase mRNA expression was increased by injury and decreased by exercise. Our results demonstrate that exercise affects expression of genes involved in plasticity and apoptosis in a cell specific manner and that these change with increased post-injury intervals and/or prolonged periods of exercise.

Activity-dependent increase in neurotrophic factors is associated with an enhanced modulation of spinal reflexes after spinal cord injury.

Activity-based therapies such as passive bicycling and step-training on a treadmill contribute to motor recovery after spinal cord injury (SCI), leading to a greater number of steps performed, improved gait kinematics, recovery of phase-dependent modulation of spinal reflexes, and prevention of decrease in muscle mass. Both tasks consist of alternating movements that rhythmically stretch and shorten hindlimb muscles. However, the paralyzed hindlimbs are passively moved by a motorized apparatus during bike-training, whereas locomotor movements during step-training are generated by spinal networks triggered by afferent feedback. Our objective was to compare the task-dependent effect of bike- and step-training after SCI on physiological measures of spinal cord plasticity in relation to changes in levels of neurotrophic factors. Thirty adult female Sprague-Dawley rats underwent complete spinal transection at a low thoracic level (T12). The rats were assigned to one of three groups: bike-training, step-training, or no training. The exercise regimen consisted of 15 min/d, 5 days/week, for 4 weeks, beginning 5 days after SCI. During a terminal experiment, H-reflexes were recorded from interosseus foot muscles following stimulation of the tibial nerve at 0.3, 5, or 10 Hz. The animals were sacrificed and the spinal cords were harvested for Western blot analysis of the expression of neurotrophic factors in the lumbar spinal cord. We provide evidence that bike- and step-training significantly increase the levels of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4 in the lumbar enlargement of SCI rats, whereas only step-training increased glial cell-derived neurotrophic factor (GDNF) levels. An increase in neurotrophic factor protein levels that positively correlated with the recovery of H-reflex frequency-dependent depression suggests a role for neurotrophic factors in reflex normalization.

Arg/Abl-binding protein, a Z-body and Z-band protein, binds sarcomeric, costameric, and signaling molecules.

ArgBP2 (Arg/Abl-Binding Protein) is expressed at high levels in the heart and is localized in the Z-bands of mature myofibrils. ArgBP2 is a member of a small family of proteins that also includes vinexin and CAP (c-Cbl-associated protein), all characterized by having one sorbin homology (SOHO) domain and three C-terminal SH3 domains. Antibodies directed against ArgBP2 also react with the Z-bodies of myofibril precursors: premyofibrils and nascent myofibrils. Expression in cardiomyocytes of plasmids encoding Yellow Fluorescent Protein (YFP) fused to either full length ArgBP2, the SOHO, mid-ArgBP or the SH3 domains of ArgBP2 led to Z-band targeting of the fusion proteins, whereas an N-terminal fragment lacking these domains did not target to Z-bands. Although ArgBP2 is not found in skeletal muscle cells, YFP-ArgBP2 did target to Z-bodies and Z-bands in cultured myotubes. GST-ArgBP2-SH3 bound actin, α-actinin and vinculin proteins in blot overlays, cosedimentation assays, and EM negative staining techniques. Over-expression of ArgBP2 and ArgBP2-SH3 domains, but not YFP alone, led to loss of myofibrils in cardiomyocytes. Fluorescence recovery after photobleaching was used to measure the rapid dynamics of both the full length and some truncated versions of ArgBP2. Our results indicate that ArgBP2 may play an important role in the assembly and maintenance of myofibrils in cardiomyocytes.

Cycling exercise affects the expression of apoptosis-associated microRNAs after spinal cord injury in rats.

There are two major aspects to a spinal cord injury (SCI): an acute, primary mechanical trauma and a progressive phase of secondary tissue damage provoked by inflammation, excitotoxicity, apoptosis, and demyelination. MicroRNAs (miRs) are small, ~22 nucleotide, non-protein-coding RNAs that function at the post-transcriptional level to regulate gene expression. They have important roles in homeostatic processes such as cell proliferation and programmed cell death. In the injured rat spinal cord we performed an expression analysis of miRs and their downstream targets involved in apoptotic pathways and used post-injury cycling exercise to test for activity-dependent plasticity of miR expression. We show that SCI results in increased expression of miR Let-7a and miR16 while exercise leads to elevated levels of miR21 and decreased levels of miR15b. These changes in miR expression are correlated with changes in expression of their target genes: pro-apoptotic (decreased PTEN, PDCD4, and RAS mRNA) and anti-apoptotic (increased Bcl-2 mRNA) target genes. This is accompanied by a down-regulation of mRNA for caspase-7 and caspase-9 and reduced levels of caspase-7 protein. These results indicate possible beneficial effects of exercise through action on multiple miRs and their targets that contribute to the functional regulation of apoptosis after SCI.

Secretion profile of human bone marrow stromal cells: donor variability and response to inflammatory stimuli.

Mesenchymal stem cells (MSC) derived from bone marrow are ideal transplants for a variety of CNS disorders and appear to support recovery after injury by secreting therapeutic factors. There is considerable variability in the secretion profile of MSC derived from different donors and it is known that MSC secretion changes in response to inflammatory stimuli, but no comprehensive analysis has been performed to address these issues. Here we show that MSC from seven donors secrete chemokines and cytokines in variable ranges, with some factors showing high variability. Treatment of cultured MSC with pro-inflammatory cytokines or tissue extracts from injured spinal cord resulted in up-regulation of selected cytokines, whereas treatment with an anti-inflammatory cytokine had little effect, indicating that the secretion profile is tightly regulated by environmental challenges. Patterns of up-regulated cytokines were similar in MSC from different donors suggesting a comparable response to inflammatory stimuli.

PEGylated interferon-beta modulates the acute inflammatory response and recovery when combined with forced exercise following cervical spinal contusion injury.

Secondary degeneration leads to an expansion of the initial tissue damage sustained during a spinal cord injury (SCI). Dampening the cellular inflammatory response that contributes to this progressive tissue damage is one possible strategy for neuroprotection after acute SCI. We initially examined whether treatment with a PEGylated form of rat interferon-beta (IFN-beta) would modulate the expression of several markers of inflammation and neuroprotection at the site of a unilateral cervical level 5 contusion injury. Adult female Sprague-Dawley rats were injured using the Infinite Horizon Impactor at a force of 200 kdyn (equivalent to a severe injury) and a mean displacement of 1600-1800 mum. A single dose (5x10(6) units) of PEGylated IFN-beta or vehicle was administered 30 min following SCI. Here we demonstrate temporal changes in pro- and anti-inflammatory cytokine levels and the expression of heat shock proteins and iNOS (involved in neuroprotection) at the lesion epicenter and one segment caudally after SCI and PEG IFN-beta treatment. The results suggested a potential therapeutic treatment strategy for modulation of secondary damage after acute SCI. Therefore, we examined whether acute treatment with PEG IFN-beta would improve forelimb function alone or when combined with forced exercise (Ex). Animals began the Ex paradigm 5 days post SCI and continued for 5 days/week over 8 weeks. Locomotion (forelimb locomotor scale [FLS], hindlimb BBB, and TreadScan) and sensorimotor function (grid walking) was tested weekly. Additional outcome measures included lesion size and glial cell reactivity. Significant FLS improvements occurred at 1 week post SCI in the PEGylated IFN-beta-treated group but not at any other time point or with any other treatment approaches. These results suggest that this acute neuroprotective treatment strategy does not translate into long term behavioral recovery even when combined with forced exercise.

Forced exercise as a rehabilitation strategy after unilateral cervical spinal cord contusion injury.

Evaluation of locomotor training after spinal cord injury (SCI) has primarily focused on hind limb recovery, with evidence of functional and molecular changes in response to exercise. Since trauma at a cervical (C) level is common in human SCI, we used a unilateral C4 contusion injury model in rats to determine whether forced exercise (Ex) would affect spinal cord biochemistry, anatomy, and recovery of fore and hind limb function. SCI was created with the Infinite Horizon spinal cord impactor device at C4 with a force of 200 Kdyne and a mean displacement of 1600-1800 microm in adult female Sprague-Dawley rats that had been acclimated to a motorized exercise wheel apparatus. Five days post-operatively, the treated group began Ex on the wheel for 20 min per day, 5 days per week for 8 weeks. Wheel speed was increased daily according to the abilities of each animal up to 14 m/min. Control rats were handled daily but were not exposed to Ex. In one set of animals experiencing 5 days of Ex, there was a moderate increase in brain-derived neurotrophic factor (BDNF) and heat shock protein-27 (HSP-27) levels in the lesion epicenter and surrounding tissue. Long-term (8 weeks) survival groups were exposed to weekly behavioral tests to assess qualitative aspects of fore limb and hind limb locomotion (fore limb scale, FLS and BBB [Basso, Beattie, and Bresnahan locomotor rating scale]), as well as sensorimotor (grid) and motor (grip) skills. Biweekly assessment of performance during wheel walking examined gross and fine motor skills. The FLS indicated a significant benefit of Ex during weeks 2-4. The BBB test showed no change with Ex at the end of the 8-week period, however hind limb grid performance was improved during weeks 2-4. Lesion size was not affected by Ex, but the presence of phagocytic and reactive glial cells was reduced with Ex as an intervention. These results suggest that Ex alone can influence the evolution of the injury and transiently improve fore and hind limb function during weeks 2-4 following a cervical SCI.

Unexpected abundance of pathological tau in progressive supranuclear palsy white matter.

OBJECTIVE: To investigate whether biochemical insoluble tau with 4 (4R) and/or 3 (3R) microtubule-binding repeats accumulate in white as well as gray matter in progressive supranuclear palsy (PSP), a neurodegenerative tauopathy. METHODS: To assess tau pathology in PSP white matter, we combined Western blot (WB) and immunohistochemical methods to analyze 23 autopsy-confirmed PSP brains. RESULTS: WBs showed an unexpected abundance of insoluble tau in white and gray matter of PSP brains, but biochemical tau pathology in white matter was not correlated with immunohistochemistry using the same panel of epitope-specific anti-tau antibodies used for WB. Despite heterogeneity in the representation of pathological 3R and 4R tau isoforms in cortical versus subcortical regions, biochemically detectable white matter tau pathology is a constant feature of PSP. INTERPRETATION: These studies show additional similarities between PSP and corticobasal degeneration, but unlike corticobasal degeneration, more abundant white matter tau pathology in PSP is detectable by WB than by immunohistochemistry. The differential detection of abnormal tau by biochemistry versus microscopy in PSP may reflect distinct pathological mechanisms, and elucidation of these processes will augment efforts to develop better strategies for the diagnosis and treatment of PSP and related neurodegenerative tauopathies.

Characterization of tau pathologies in gray and white matter of Guam parkinsonism-dementia complex.

Guam parkinsonism-dementia complex (PDC) is a neurodegenerative tauopathy in ethnic Chamorro residents of the Mariana Islands that manifests clinically with parkinsonism as well as dementia and is characterized neuropathologically by prominent cortical neuron loss in association with extensive telencephalic neurofibrillary tau pathology. To further characterize cortical gray and white matter tau, alpha-synuclein and lipid peroxidation pathologies in Guam PDC, we examined the brains of 17 Chamorro PDC and control subjects using biochemical and immunohistological techniques. We observed insoluble tau pathology in both gray and white matter of PDC and Guam control cases, with frontal and temporal lobes being most severely affected. Using phosphorylation dependent anti-tau antibodies, abundant tau inclusions were detected by immunohistochemistry in both neuronal and glial cells of the neocortex, while less alpha-synuclein pathology was observed in more limited brain regions. Further, in sharp contrast to Alzheimer's disease (AD), levels of the lipid peroxidation product 8, 12-iso-iPF(2alpha)-VI isoprostane were not elevated in Guam PDC brains relative to controls. Thus, although the tau pathologies of Guam PDC share similarities with AD, the composite Guam PDC neuropathology profile of tau, alpha-synuclein and 8, 12-iso-iPF(2alpha)-VI isoprostane reported here more closely resembles that seen in other tauopathies including frontotemporal dementias (FTDs), which may imply that Guam PDC and FTD tauopathies share underlying mechanisms of neurodegeneration.

alpha-internexin is present in the pathological inclusions of neuronal intermediate filament inclusion disease.

Neuronal intermediate filament (IF) inclusion disease (NIFID) is a novel neurological disease of early onset with a variable clinical phenotype including frontotemporal dementia, pyramidal, and extrapyramidal signs. Pathologically, in affected areas, there is neuronal loss, astrocytosis, and neuronal intracytoplasmic aggregates of abnormal neuronal IFs that contain neither tau nor alpha-synuclein. Thus, to characterize the neuronal IF protein profile of inclusions in NIFID, immunohistochemistry (IHC) was performed on 10 cases of NIFID, four normal aged controls (NL), and two cases of Alzheimer's disease (AD) using a panel of anti-neuronal IF proteins. Immunoelectron microscopy was performed on selected cases and frozen tissue from the frontal lobe of four cases was used for biochemical studies including sequential extractions and Western blotting. Based on these studies, we report here for the first time that alpha-internexin, a neuronal IF protein, is present within the inclusions of NIFID as are all three neurofilament subunits: heavy, medium, and light. Thus, all class IV neuronal IF proteins are present within the pathological inclusions of this disease. Biochemistry revealed that IF aggregates were soluble in sodium dodecyl sulfate (SDS) and no post-translational modification was detected when compared with Alzheimer's disease or aged control brains. Hence, we conclude that NIFID is characterized by the pathological cytoplasmic aggregation of all class IV neuronal IF proteins in brain. The discovery of alpha-internexin in the cytoplasmic inclusions implicates novel mechanisms of pathogenesis in NIFID and other neurological diseases with pathological accumulations of IFs.

12/15-lipoxygenase is increased in Alzheimer's disease: possible involvement in brain oxidative stress.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that impairs cognition and behavior. Although the initiating molecular events are not known, increasing evidence suggests that oxidative stress could play a functional role in its pathogenesis. Lipoxygenase (LOX) enzymes by oxidizing polyunsaturated fatty acids synthesize hydroperoxyacids, which are potent pro-oxidant mediators. Because circumstantial evidence suggests that 12/15-LOX is a major source of oxidative stress, we investigated the protein levels and activity of this enzyme in different brain regions of histopathologically confirmed AD and control cases. Using quantitative Western blot analysis we demonstrated that in affected frontal and temporal regions of AD brains the amount of 12/15-LOX was higher compared with controls, whereas no difference between the two groups was detected in the cerebellum. This observation was confirmed by immunohistochemical studies. Levels of 12/15-hydroxyeicosatetraenoic acids, metabolic products of 12/15-LOX, were also markedly elevated in AD brains compared to controls. This increase directly correlated with brain lipid peroxidation, and inversely with vitamin E levels. Finally, genetic deletion of this enzyme in vitro resulted in a reduction of the cellular oxidative stress response after incubation with H2O2 or amyloid beta. These data show that the 12/15-LOX metabolic pathway is increased and correlates with an oxidative imbalance in the AD brain, implying that this enzyme might contribute to the pathogenesis of this neurodegenerative disorder.

Assessment of pathological tau proteins in frontotemporal dementias: qualitative and quantitative approaches.

A large group of clinically and neuropathologically heterogeneous frontotemporal dementias (FTDs) is distinguished by the accumulation of filamentous tau proteins in neuronal and non-neuronal cells, and these disorders are collectively designated as tauopathies. Recent advances in the development of new reagents and the utilization of new multidisciplinary approaches have made substantial progress in the extensive characterization of tau proteins in different tauopathies. Until recently, semi-quantitative assessment of specific pathological lesions in FTDs was limited to immunohistochemical methods. However, it has become increasingly evident that biochemical analyses of pathological tau proteins in diseased brains also play a significant role in establishing the diagnosis of specific FTD subtypes. In this review, the authors summarize recent findings of the unique biochemical tau signatures for a number of specific tauopathies. Moreover, the quantitative assessment of abnormal tau proteins described here reveals heterogeneity in the biochemical features of tau pathologies in FTDs. The identification of highly insoluble tau species that may be more prone to physiological clearance, other than those that form paired helical filaments, could provide additional insights into the role of tau in mechanisms of neurodegeneration in FTDs.

DJ-1 colocalizes with tau inclusions: a link between parkinsonism and dementia.

Two novel mutations recently have been identified in the DJ-1 gene that cause a new form of autosomal recessive, early-onset parkinsonism. Because the pathological role of this protein is unknown, we examined the issue here and report the colocalization of DJ-1 protein within a subset of pathological tau inclusions in a diverse group of neurodegenerative disorders known as tauopathies. Our study extends the view that different neurodegenerative diseases may have similar pathological mechanisms, and that these processes likely include DJ-1.

Molecular identification of AMY, an Alzheimer disease amyloid-associated protein.

One of the neuropathological lesions characteristic of Alzheimer disease (AD) is the cerebral accumulation of the amyloid beta-peptide (A beta). Although numerous studies have demonstrated that A beta spontaneously forms amyloid in vitro, the molecular events underlying A beta amyloid formation in vivo are less well understood. Immunohistochemical studies have shown that other proteins colocalize with A beta in amyloid deposits in brain. The identity of one of these proteins, AMY, has so far remained elusive; therefore we attempted to purify AMY. The AMY protein was found to co-purify with A beta in insoluble fractions from human AD brain, and was absent in brains from control subjects. AMY immunoreactivity was primarily restricted to a 50-kDa and 100-kDa protein species. Interestingly, the chromatographic and immunological profile of AMY resembled the recently identified amyloid-associated protein CLAC, derived from a transmembrane collagen-like precursor, CLAC-P. Antibodies against AMY recognized CLAC-P expressed in mammalian cells. In addition, side-by-side comparisons of AD brain sections and extracts, using antibodies against both AMY and CLAC, respectively, resulted in almost identical staining patterns. Therefore, we conclude that the AMY immunoreactivity seen in association with amyloid in AD brain is due to the presence of the CLAC protein.