Deng Shun Wang

Comparison of kindreds with parkinsonism and α‐synuclein genomic multiplications

Genomic triplication of the α‐synuclein gene recently has been associated with familial Parkinsons disease in the Spellman–Muenter kindred. Here, we present an independent family, of Swedish‐American descent, with hereditary early‐onset parkinsonism with dementia due to α‐synuclein triplication. Brain tissue available from affected individuals in both kindreds provided the opportunity to compare their clinical, pathological, and biochemical phenotypes. Of note, studies of brain mRNA and soluble protein levels demonstrate a doubling of α‐synuclein expression, consistent with molecular genetic data. Pathologically, cornu ammonis 2/3 hippocampal neuronal loss appears to be a defining feature of this form of inherited parkinsonism. The profound implications of α‐synuclein overexpression for idiopathic synucleinopathies are discussed.

β-Amyloid degradation and Alzheimer's disease

Extensive β-amyloid (Aβ) deposits in brain parenchyma in the form of senile plaques and in blood vessels in the form of amyloid angiopathy are pathological hallmarks of Alzheimers disease (AD). The mechanisms underlying Aβ deposition remain unclear. Major efforts have focused on Aβ production, but there is little to suggest that increased production of Aβ plays a role in Aβ deposition, except for rare familial forms of AD. Thus, other mechanisms must be involved in the accumulation of Aβ in AD. Recent data shows that impaired clearance may play an important role in Aβ accumulation in the pathogenesis of AD. This review focuses on our current knowledge of Aβ-degrading enzymes, including neprilysin (NEP), endothelin-converting enzyme (ECE), insulin-degrading enzyme (IDE), angiotensin-converting enzyme (ACE), and the plasmin/uPA/tPA system as they relate to amyloid deposition in AD.

Expression and functional profiling of neprilysin, insulin‐degrading enzyme, and endothelin‐converting enzyme in prospectively studied elderly and Alzheimer’s brain

J. Neurochem. (2010) 115, 47–57.

The significance of Pin1 in the development of Alzheimer's disease.

Pin1 protein, a peptidyl-prolyl cis-trans isomerase plays an important regulatory role in neuronal function. Recent studies indicate that Pin1 may promote the dephosphorylation of tau and restore its ability to bind to and polymerize microtubles. Previous studies on postmortem human brains showed that Pin1 is down-regulated in advanced Alzheimers disease (AD) brains compared to age-matched non-demented controls. Because AD is a slowly progressive disease with a preclinical period that can last years, the abundance and regulatory function of Pin1 may vary on the course of the disease. In order to evaluate the potential contribution of Pin1 to AD pathogenesis, levels of mRNA, protein and isomerase activity of Pin1 and phosphorylated tau from postmortem brains of 10 persons with mild-cognitive impairment (MCI), 10 with AD and 10 age-matched no cognitive impairment (NCI) were measured. The relationship between Pin1 and phosphorylated tau as well as clinical and cognitive data were analyzed. The results indicated that Pin1 activity in MCI and AD were significantly higher than in NCI. Phosphorylated tau in MCI and AD was also higher than in NCI group. The positive correlation trend in MCI and the robust correlation in AD between Pin1 activity and phosphorylated tau implies that increasing phosphorylated tau during AD pathogenesis may induce the compensatory activation/up-regulation of Pin1, while the inverse correlation between Pin1 activity and phosphorylated tau in NCI group implies that decreased Pin1 may play a role in the initial accumulation of phosphorylated tau in AD pathogenesis.

Effects of HNE‐modification induced by Aβ on neprilysin expression and activity in SH‐SY5Y cells

The cerebral accumulation of β‐amyloid (Aβ) is a consistent feature of and likely contributor to the development of Alzheimer’s disease. In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism also plays an important role in Aβ accumulation. We have previously shown that neprilysin (NEP), the major protease which cleaves Aβin vivo, is modified by 4‐hydroxy‐nonenal (HNE) adducts in the brain of Alzheimer’s disease patients. To determine if these changes affected Aβ, SH‐SY5Y cells were treated with HNE or Aβ, and then NEP mRNA, protein levels, HNE adducted NEP, NEP activity and secreted Aβ levels were determined. Intracellular NEP developed HNE adducts after 24 h of HNE treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. HNE‐modified NEP showed decreased catalytic activity, which was associated with elevations in Aβ1–40 in SH‐SY5Y and H4 APP695wt cells. Incubation of cells with Aβ1–42 also induced HNE adduction of NEP. In an apparent compensatory response, Aβ‐treated cells showed increased NEP mRNA and protein expression. Despite elevations in NEP protein, the activity was significantly lower compared with the NEP protein level. This study demonstrates that NEP can be inactivated by HNE‐adduction, which is associated with, at least partly, reduced Aβ cleavage and enhanced Aβ accumulation.

Screening for neurofilament inclusion disease using α-internexin immunohistochemistry

Neurofilament inclusion disease (NFID) is characterized by cortical and subcortical neuronal loss and neuronal cytoplasmic inclusions composed of neuronal intermediate filament proteins.1–6 Almost all cases have presented with a syndrome typical of a frontotemporal lobar degeneration (FTLD).1–6 More recently, the clinical heterogeneity of NFID has been emphasized, with some cases presenting with parkinsonism or motor neuron disease,1 including cases resembling ALS or primary lateral sclerosis (PLS). Several cases have also had clinical and pathologic features that overlap with multiple-system atrophy (MSA).1–6 Whereas the neuronal filamentous inclusions in NFID are immunoreactive with neurofilament (NF) antibodies and negative for tau and α-synuclein,1–6 α-internexin may be the most sensitive marker for these particular inclusions.1–3 Given this observation, we investigated the frequency of NFID using α-internexin immunohistochemistry in 92 cases that had been diagnosed neuropathologically as FTLD, ALS, PLS, or MSA but in which routine screening with immunohistochemistry for NF had not been previously done. A consecutive series of 95 cases of diverse neurodegenerative diseases, including 3 cases of NFID, from the neuropathology files of Mayo Clinic …

Decreases in soluble α-synuclein in frontal cortex correlate with cognitive decline in the elderly

Alpha-synuclein (ASN) is a presynaptic protein and major component of Lewy bodies. It is considered important in the pathogenesis of Alzheimers disease (AD), but its relevance to progressive cognitive decline in aging is largely unknown. To address this issue, ASN immunoreactivity was measured in frontal cortex of elderly individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and early AD using a Western blot technique and a polyclonal antibody to ASN. ASN immunoreactivity was significantly lower in AD than in MCI and NCI, but there was no difference between MCI and NCI. The ASN immunoreactivity correlated with CERAD diagnosis, as well as Mini-Mental State Exam (MMSE) score, global neuropsychologic z-score and some, but not all, frontal neuropsychology measures. Clinical correlations were stronger for ASN than synaptophysin immunoreactivity assessed in a similar manner. The correlation with MMSE was robust when NCI cases were considered separately, suggesting that decreases in soluble ASN may be an early feature of cognitive decline in aging and AD.

Cognitive Performance Correlates with Cortical Isopeptide Immunoreactivity as Well as Alzheimer Type Pathology

BACKGROUND Protein cross-linking and aggregation are important molecular processes in Alzheimers disease (AD), and tissue transglutaminase (tTG) catalyzes protein cross-linking. OBJECTIVES To measure tTG, tTG enzyme activity and isopeptide, which is the product of tTG, in brain and to relate them to cognitive scores. METHODS tTG and isopeptide levels were measured in frontal gray matter of 10 normal (NCI), 10 mild cognitive impairment (MCI) and 9 AD brains from the Religious Orders Study. tTG enzymatic activity was measured with a fluorescence assay. RESULTS tTG protein and enzyme activity were highest in AD, but not significantly greater than MCI or NCI. In contrast, isopeptide immunoreactivity in formic acid extracts was significantly greater in AD than NCI and MCI. The level of insoluble formic acid extractable isopeptide correlated with several measures of cognitive function, including word generation and perceptual speed. Multiple linear regression analyses indicated that insoluble isopeptide immunoreactivity could be accounted for by a combination of factors in the formic acid extract, including Abeta, ubiquitin and tau. CONCLUSIONS Accumulation of insoluble proteins with isopeptide bonds correlates with cognitive impairment. The relationship of isopeptide to other proteins that are also enriched in formic acid extracts suggests that several substrates of tTG may play a role in the pathogenesis of AD.

N-Acetylcysteine Prevents 4-Hydroxynonenal- and Amyloid-β-Induced Modification and Inactivation of Neprilysin in SH-SY5Y Cells

As one of the dominant amyloid-beta peptide (Abeta) proteases, neprilysin (NEP) plays a crucial role in maintaining a physiologic balance between Abeta production and catabolism. We have previously shown that NEP is modified by 4-hydroxynonenal (HNE) adducts, resulting in decreased activity in the brain of AD patients and cultured cells. In order to determine whether antioxidants can rescue NEP, SH-SY5Y cells were treated with HNE or Abeta, together with N-acetylcysteine for 24 hours, prior to analysis of NEP protein levels, activity, and oxidative modifications. Intracellular NEP developed HNE adducts after 24 hours of HNE or Abeta treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. N-acetylcysteine at 10 to 100 microM alleviated HNE adduction after HNE or Abeta treatment. In keeping with previous reports, HNE-modified NEP showed decreased catalytic activity. The present study demonstrates that antioxidants can be used to spare NEP from oxidative modification, suggesting a potential mechanism underlying the neuroprotective effects of antioxidants in aging or Alzheimers disease.

Effects of 4-Hydroxy-Nonenal and Amyloid-β on Expression and Activity of Endothelin Converting Enzyme and Insulin Degrading Enzyme in SH-SY5Y Cells

The cerebral accumulation of amyloid-beta (Abeta) is a consistent feature of and likely contributor to the development of Alzheimers disease (AD). In addition to dysregulated production, increasing experimental evidence suggests reduced catabolism plays an important role in Abeta accumulation. Although endothelin converting enzyme (ECE) and insulin degrading enzyme (IDE) degrade and thus contribute to regulating the steady-state levels of Abeta, how these enzymes are regulated remain poorly understood. In this study, we investigated the effects of 4-hydroxy-nonenal (HNE) and Abeta on the expression and activity of ECE-1 and IDE in human neuroblastoma SH-SY5Y cells. Treatment with HNE or Abeta upregulated ECE-1 mRNA and protein, while IDE was unchanged. Although both ECE-1 and IDE were oxidized within 24 h of HNE or Abeta treatment, ECE-1 catalytic activity was elevated while IDE specific activity was unchanged. The results demonstrated for the first time that both ECE-1 and IDE are substrates of HNE modification induced by Abeta. In addition, the results suggest complex mechanisms underlying the regulation of their enzymatic activity.