Howard D. Rees

Oxidative Modifications and Down-regulation of Ubiquitin Carboxyl-terminal Hydrolase L1 Associated with Idiopathic Parkinson's and Alzheimer's Diseases

Alzheimers disease (AD) and Parkinsons 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.

Oxidative Damage of DJ-1 Is Linked to Sporadic Parkinson and Alzheimer Diseases

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.

Familial Parkinson's Disease-associated L166P Mutation Disrupts DJ-1 Protein Folding and Function

Mutations in DJ-1, a protein of unknown function, were recently identified as the cause for an autosomal recessive, early onset form of familial Parkinsons 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 Parkinsons 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 Parkinsons disease.

Immunohistochemical localization of subtype 4a metabotropic glutamate receptors in the rat and mouse basal ganglia

Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild‐type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a‐transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g‐amino‐n‐butyric acid (GABA) release from striatopallidal terminals. J. Comp. Neurol. 407:33–46, 1999.

Polyubiquitin Linkage Profiles in Three Models of Proteolytic Stress Suggest the Etiology of Alzheimer Disease

Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys48), 38% (Lys63), 8% (Lys29), 2% (Lys11), and 0.5% or less for linear, Lys6, Lys27, and Lys33 linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys6, Lys11, Lys27, Lys29, and Lys48 mediate proteasomal degradation, whereas Lys63 chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys48, may also contribute to lysosomal targeting, as both Lys63 and Lys48 linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys11, Lys48, and Lys63 but not Lys29 linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease

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 box-dependent-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.

Neuronal LR11/SorLA Expression Is Reduced in Mild Cognitive Impairment

LR11 (aka sorLA) is a multifunctional neuronal receptor that binds apolipoprotein E and interacts with amyloid precursor protein to regulate amyloidogenesis. Reduced expression of LR11, as occurs in the brains of individuals with Alzheimers disease (AD), increases amyloidogenesis, and variants in the gene that encodes LR11, SORL1, have recently been linked to risk for late‐onset AD. In this study, we sought to determine whether reduced expression of LR11 occurs early in the disease process and whether protein levels in cortical neurons are associated with clinical and pathological changes in mild cognitive impairment (MCI), a condition that may represent prodromal AD.

Distribution and Developmental Regulation of Metabotropic Glutamate Receptor 7a in Rat Brain

Abstract: To determine the regional and cellular distribution of the metabotropic glutamate receptor mGluR7a, we used rabbit anti‐peptide polyclonal‐targeted antibodies against the C‐terminal domain of mGluR7a. Here we report that immunocytochemistry at the light‐microscopic level revealed that mGluR7a is widely distributed throughout the adult rat brain, with a high level of expression in sensory areas, such as piriform cortex, superior colliculus, and dorsal cochlear nucleus. In most brain structures, mGluR7a immunoreactivity is characterized by staining of puncta and fibers. However, in some regions, including the locus ceruleus, cerebellum, and thalamic nuclei, both cell bodies and fibers are immunopositive. The changes in levels of mGluR7a during development were investigated with immunoblotting and immunocytochemical analysis. Immunoblot analysis revealed that the levels of mGluR7a are differentially regulated across brain regions during postnatal development. In cortical regions (hippocampus, neocortex, and olfactory cortex), mGluR7a levels were highest at postnatal day 7 (P7) and P14, then declined in older rats. In contrast, mGluR7a levels were highest at P7 in pons/medulla and cerebellum and decreased markedly between P7 and P14. In these regions, mGluR7a immunoreactivity was at similar low levels at P14 and P21 and in adults. Immunocytochemical analysis revealed that staining for mGluR7a was exceptionally high in fiber tracts in P7 animals relative to adults. Furthermore, the pattern of mGluR7a immunoreactivity in certain brain structures, including cerebellum, piriform cortex, and hippocampus, was significantly different in P7 and adult animals. In summary, these data suggest that mGluR7a is widely distributed throughout the rat brain and that this receptor undergoes a dynamic, regionally specific regulation during postnatal development.

Interaction of Akt-phosphorylated SRPK2 with 14-3-3 Mediates Cell Cycle and Cell Death in Neurons

Terminally differentiated neurons are unable to reenter the cell cycle. Aberrant cell cycle activation provokes neuronal cell death, whereas cell cycle inhibition elevates neuronal survival. However, the molecular mechanism regulating the cell cycle and cell death in mature neurons remains elusive. Here we show that SRPK2, a protein kinase specific for the serine/arginine (SR) family of splicing factors, triggers cell cycle progression in neurons and induces apoptosis through regulation of nuclear cyclin D1. Akt phosphorylates SRPK2 on Thr-492 and promotes its nuclear translocation leading to cyclin D1 up-regulation, cell cycle reentry, and neuronal apoptosis. In addition, SRPK2 phosphorylates SC35 and, thus, inactivates p53, resulting in cyclin D1 up-regulation. 14-3-3 binding to SRPK2, regulated by Akt phosphorylation, inhibits these events. We find that SRPK2 is phosphorylated in ischemia-attacked brain, correlating with the observed increase in cyclin D1 levels. Hence, phosphatidylinositol 3-kinase/Akt mediates the cell cycle and cell death machinery in the nervous system through phosphorylation of SRPK2.

Autoradiographic studies of 3H-dexamethasone uptake by immunocytochemically characterized cells of the rat pituitary.

Summary3H-Dexamethasone (10 μg/kg) was injected intravenously in adrenalectomized rats and after survival times of 5, 30, 60, and 180 min its uptake within the pituitary was studied by autoradiography. Radioactivity was concentrated in cell nuclei in the pars nervosa and pars distalis. Within the pars intermedia, only cells of the marginal zone were labeled. In the pars distalis, some cells showed a weak nuclear accumulation of radioactivity as early as 5 min after injection. The tissue radioactivity was nearly maximal at 5 min, and the proportion of radioactivity in nuclei reached a maximum of 60–70% by 30 min.In competition experiments, non-radioactive steroids (1 mg/kg) were injected 5 min before 3H-dexamethasone and sacrifice was 30 min later. Dexamethasone markedly diminished the nuclear accumulation in the pars distalis, but corticosterone and progesterone did not. In the pars nervosa, corticosterone and progesterone competed for nuclear uptake of 3H-dexamethasone, although less effectively than dexamethasone itself.Different cell types in the pars distalis were characterized by treating autoradiograms with an immuno-peroxidase bridge procedure. Cells treated with anti-ACTH 17–39 had the greatest nuclear concentration of radioactivity, and those stained with anti-TSH were least heavily labeled. Cells treated with antisera to GH, PRL, and hCG were moderately labeled.