Prosthetic devices that are controlled by intracortical electrodes recording one's 'thoughts' are a reality today, and no longer merely in the realm of science fiction. However, widespread clinical use of implanted electrodes is hampered by a lack of reliability in chronic recordings, independent of the type of electrodes used. One major hypothesis has been that astroglial scar electrically impedes the electrodes. However, there is a temporal discrepancy between stabilization of scar's electrical properties and recording failure with recording failure lagging by 1 month. In this study, we test a possible explanation for this discrepancy: the hypothesis that chronic inflammation, due to the persistent presence of the electrode, causes a local neurodegenerative state in the immediate vicinity of the electrode. Through modulation of chronic inflammation via stab wound, electrode geometry and age-matched control, we found that after 16 weeks, animals with an increased level of chronic inflammation were associated with increased neuronal and dendritic, but not axonal, loss. We observed increased neuronal and dendritic loss 16 weeks after implantation compared to 8 weeks after implantation, suggesting that the local neurodegenerative state is progressive. After 16 weeks, we observed axonal pathology in the form of hyperphosphorylation of the protein tau in the immediate vicinity of the microelectrodes (as observed in Alzheimer's disease and other tauopathies). The results of this study suggest that a local, late onset neurodegenerative disease-like state surrounds the chronic electrodes and is a potential cause for chronic recording failure. These results also inform strategies to enhance our capability to attain reliable long-term recordings from implantable electrodes in the CNS.

Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc boxdependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein.Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.proteomics | liquid chromatography-tandem mass spectrometry | U1A | RNA-seq | premature cleavage and polyadenylation

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that occur either in relatively rare, familial forms or in common, sporadic forms. The genetic defects underlying several monogenic familial forms of AD and PD have recently been identified, however, the causes of other AD and PD cases, particularly sporadic cases, remain unclear. To gain insights into the pathogenic mechanisms involved in AD and PD, we used a proteomic approach to identify proteins with altered expression levels and/or oxidative modifications in idiopathic AD and PD brains. Here, we report that the protein level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a neuronal de-ubiquitinating enzyme whose mutation has been linked to an early-onset familial PD, is down-regulated in idiopathic PD as well as AD brains. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified three human brain UCH-L1 isoforms, a full-length form and two amino-terminally truncated forms. Our proteomic analyses reveal that the full-length UCH-L1 is a major target of oxidative damage in AD and PD brains, which is extensively modified by carbonyl formation, methionine oxidation, and cysteine oxidation. Furthermore, immunohistochemical studies show that prominent UCH-L1 immunostaining is associated with neurofibrillary tangles and that the level of soluble UCH-L1 protein is inversely proportional to the number of tangles in AD brains. Together, these results provide evidence supporting a direct link between oxidative damage to the neuronal ubiquitination/de-ubiquitination machinery and the pathogenesis of sporadic AD and PD.Alzheimer's disease (AD) 1 and Parkinson's disease (PD) are the two most common neurodegenerative disorders in humans, however, the causes of AD and PD, particularly the sporadic cases, remain unclear. A prominent feature of AD and PD as well as other neurodegenerative diseases is the accumulation of insoluble proteinaceous deposits, such as senile plaques and neurofibrillary tangles in AD and Lewy bodies in PD (1). Although these deposits have different protein compositions, they all contain ubiquitin and ubiquitinated proteins (2). Interestingly, mutations in two enzymes of the ubiquitination/de-ubiquitination system, parkin and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), have been identified as causative genetic defects for certain familial forms of PD (3). In familial AD patients, an aberrant form of ubiquitin resulting from a ϩ1 frameshift mutation in the ubiquitin-B gene has been detected (4). Moreover, impaired proteasome function has been reported in idiopathic AD and PD brains (2). Collectively, these different lines of evidence support a role for dysfunction of the ubiquitinproteasome pathway in the pathogenesis of AD and PD.Oxidative stress is another important factor that has been implicated in the pathogenesis of a number of age-related neurodegenerative diseases, including AD and PD (5, 6). Both AD and PD have been associated wit...

The precise localization of Dl and D2 dopamine receptors within striatal neurons and circuits is crucial information for further understanding dopamine pharmacology. We have used subtype specific polyclonal and monoclonal antibodies against Dl and D2 dopamine receptors to determine their cellular and subcellular distributions, their colocalization, and their differential connectivity with motor cortical afferents labeled either by lesion-induced degeneration or by anterograde transport of biotinylated dextrans. Dl and D2 are primarily expressed in medium-sized neurons and spiny dendrites. Axon terminals containing Dl were rare whereas DS-immunoreactive axon terminals forming symmetrical synapses with dendrites and spines were common. In 2 p.m sections, Dl was localized to 53% of neurons, and D2 to 48% of neurons, while mixing Dl and D2 antibodies labeled 78%. By electron microscopy, Dl was localized to 43% of dendrites and 38% of spines while D2 was localized to 38% of dendrites and 48% of spines. Combining Dl and D2 antibodies resulted in the labeling of 88.5% of dendrites and 92.6% of spines. Using different chromogens for Dl and D2, colocalization was not observed. lpsilateral motor corticostriatal afferents were primarily axospinous and significantly more synapsed with Dl than DS-positive spines (65% vs 47%). Contralateral motor corticostriatal afferents were frequently axodendritic and no difference in their frequency of synapses with Dl and D2 dendrites and spines was observed. These findings demonstrate differential patterns of expression of Dl and D2 receptorsin striatal neurons and axon terminals and their differential involvement in motor corticostriatal circuits.[

Mutations in DJ-1 cause an autosomal recessive, early onset familial form of Parkinson disease (PD). However, little is presently known about the role of DJ-1 in the more common sporadic form of PD and in other age-related neurodegenerative diseases, such as Alzheimer disease (AD). Here we report that DJ-1 is oxidatively damaged in the brains of patients with idiopathic PD and AD. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified 10 different DJ-1 isoforms, of which the acidic isoforms (pI 5.5 and 5.7) of DJ-1 monomer and the basic isoforms (pI 8.0 and 8.4) of SDS-resistant DJ-1 dimer are selectively accumulated in PD and AD frontal cortex tissues compared with age-matched controls. Quantitative Western blot analysis shows that the total level of DJ-1 protein is significantly increased in PD and AD brains. Mass spectrometry analyses reveal that DJ-1 is not only susceptible to cysteine oxidation but also to previously unsuspected methionine oxidation. Furthermore, we show that DJ-1 protein is irreversibly oxidized by carbonylation as well as by methionine oxidation to methionine sulfone in PD and AD. Our study provides new insights into the oxidative modifications of DJ-1 and indicates association of oxidative damage to DJ-1 with sporadic PD and AD.Alzheimer disease (AD) 2 and Parkinson disease (PD) are the two most common neurodegenerative disorders characterized by the selective loss of neurons in specific brain regions and the deposition of misfolded proteins into aggregates or inclusions, such as neurofibrillary tangles and amyloid plaques in AD and Lewy bodies in PD (1). The majority of AD and PD cases are sporadic with hereditary familial cases accounting for less than 10% (2, 3). The genetic defects underlying several monogenic familial forms of AD and PD have recently been identified (3). However, the causes of other AD and PD cases, particularly sporadic cases, remain unclear.Increasing evidence indicates that oxidative stress plays a crucial role in the pathogenesis of idiopathic AD and PD (4 -7). For example, both AD and PD have been associated with increased production of reactive oxygen species (ROS), which could result from a combination of aging, genetic predisposition, and environmental factors (6). Epidemiological studies suggest that exposure to pesticides, herbicides, and other environmental toxins that inhibit mitochondrial complex I can lead to excess production of ROS and increased incidence of sporadic PD (8). Furthermore, post-mortem analyses reveal that the overall levels of oxidative damage to proteins, lipids, and DNA are elevated in AD and PD brains (4, 9).The most widely used marker for oxidative damage to proteins is the presence of carbonyl groups, which can be introduced into proteins by direct oxidation of Pro, Arg, Lys, or Thr side chains or by Michael addition reactions of Cys, His, or Lys residues with products of lipid peroxidation or glycooxidation (5, 10, 11). Elevation in the total level of protein carbonyls has been doc...

Mutations in DJ-1, a protein of unknown function, were recently identified as the cause for an autosomal recessive, early onset form of familial Parkinson's disease. Here we report that DJ-1 is a dimeric protein that exhibits protease activity but no chaperone activity. The protease activity was abolished by mutation of Cys-106 to Ala, suggesting that DJ-1 functions as a cysteine protease. Our studies revealed that the Parkinson's disease-linked L166P mutation impaired the intrinsic folding propensity of DJ-1 protein, resulting in a spontaneously unfolded structure that was incapable of forming a homodimer with itself or a heterodimer with wild-type DJ-1. Correlating with the disruption of DJ-1 structure, the L166P mutation abolished the catalytic function of DJ-1. Furthermore, as a result of protein misfolding, the L166P mutant DJ-1 was selectively polyubiquitinated and rapidly degraded by the proteasome. Together these findings provide insights into the molecular mechanism by which loss-of-function mutations in DJ-1 lead to Parkinson's disease.

The presence of amyloid plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). We report here a comprehensive proteomic analysis of senile plaques from postmortem AD brain tissues. Senile plaques labeled with thioflavin-S were procured by laser capture microdissection, and their protein components were analyzed by liquid chromatography coupled with tandem mass spectrometry. We identified a total of 488 proteins coisolated with the plaques, and we found multiple phosphorylation sites on the neurofilament intermediate chain, implicating the complexity and diversity of cellular processes involved in the plaque formation. More significantly, we identified 26 proteins enriched in the plaques of two AD cases by quantitative comparison with surrounding non-plaque tissues. The localization of several proteins in the plaques was further confirmed by the approach of immunohistochemistry. In addition to previously identified plaque constituents, we discovered novel association of dynein heavy chain with the plaques in human postmortem brain and in a double transgenic AD mouse model, suggesting that neuronal transport may play a role in neuritic degeneration. Overall, our results revealed for the first time the sub-proteome of amyloid plaques that is important for further studies on disease biomarker identification and molecular mechanisms of AD pathogenesis. Alzheimer's disease (AD)1 is a devastating neurological disorder that impairs cognitive function and disturbs emotion and personality. Histopathologically, AD is manifested by the extracellular aggregation of amyloid plaques and the intraneuronal neurofibrillary tangles. Although current amyloid cascade hypothesis (1) or tau hypothesis (2) provides a framework for studying AD pathogenesis, the detailed molecular mechanisms that translate amyloid or tau accumulation into neuronal damage and functional brain impairments are largely unknown. In addition, there are numerous, complex pathological changes in AD brain that contribute to neuronal and synaptic degeneration, including mitochondrial dysfunction, oxidative damage, and inflammation (3-5).The first major breakthrough in understanding the molecular pathogenesis of AD came from the biochemical purification of amyloid ␤-peptide (A␤) from senile plaques, as described by Glenner and Wong (6), and the subsequent sequencing and identification of the A␤ precursor protein (APP) gene. Although the major insoluble component of plaques has been identified as A␤ (6, 7), the entire molecular composition of the plaques is not known. The plaques are highly complex structures with a variety of neural and glial elements (8), and many proteins have been localized to these structures by immunohistochemistry. However, biochemical verification of the plaque components has been scarce. Moreover, biochemical approaches previously applied to purify plaque components generally use very stringent extraction conditions (e.g. high concentration of salt, urea, and/or protease treatment) that may remove A␤-associat...

Background: Genetic, epidemiologic, and biochemical evidence suggests that apolipoprotein E, lowdensity lipoprotein receptors, and lipid metabolism play important roles in sporadic Alzheimer disease (AD). Objective: To identify novel candidate genes associated with sporadic AD. Design: We performed an unbiased microarray screen for genes differentially expressed in lymphoblasts of patients with sporadic AD and prioritized 1 gene product for further characterization in AD brain.