Christa L. Hladik

DNA polymerase κ deficiency does not affect somatic hypermutation in mice

Somatic hypermutation (SH) in B cells undergoing T cell‐dependent immune responses generates high‐affinity antibodies that provide protective immunity. Most current models of SH postulate the introduction of a nick into the DNA and subsequent replication‐independent, error‐prone short‐patch synthesis by one or more DNA polymerases. The PolK (DinB1) gene encodes a specialized mammalian DNA polymerase called DNA polymerase κ (polκ), a member of the recently discovered Y family of DNA polymerases. The mouse PolK gene is expressed at high levels in the seminiferous tubules of the testis and in the adrenal cortex, and at lower levels in most other cells of the body including B lymphocytes. In vitro studies showed that polκ can act as an error‐prone polymerase, although they failed to ascribe a clear function to this enzyme. The ability of polκ to generate mutations when extending primers on undamaged DNA templates identifies this enzyme as a potential candidate for the introduction of nucleotide changes in the immunoglobulin (Ig) genes during the process of SH. Here we show that polκ‐deficient mice are viable, fertile and able to mount a normal immune response to the antigen (4‐hydroxy‐3‐nitrophenyl)acetyl‐chicken γ‐globulin (NP‐GC). They also mutate their Ig genes normally. However, polκ‐deficientembryonic fibroblasts are abnormally sensitive to killing following exposure to ultraviolet (UV) radiation, suggesting a role of polκ in translesion DNA synthesis.

Olfactory bulb α-synucleinopathy has high specificity and sensitivity for Lewy body disorders

Involvement of the olfactory bulb by Lewy-type α-synucleinopathy (LTS) is known to occur at an early stage of Parkinson’s disease (PD) and Lewy body disorders and is therefore of potential usefulness diagnostically. An accurate estimate of the specificity and sensitivity of this change has not previously been available. We performed immunohistochemical α-synuclein staining of the olfactory bulb in 328 deceased individuals. All cases had received an initial neuropathological examination that included α-synuclein immunohistochemical staining on sections from brainstem, limbic and neocortical regions, but excluded olfactory bulb. These cases had been classified based on their clinical characteristics and brain regional distribution and density of LTS, as PD, dementia with Lewy bodies (DLB), Alzheimer’s disease with LTS (ADLS), Alzheimer’s disease without LTS (ADNLS), incidental Lewy body disease (ILBD) and elderly control subjects. The numbers of cases found to be positive and negative, respectively, for olfactory bulb LTS were: PD 55/3; DLB 34/1; ADLS 37/5; ADNLS 19/84; ILBD 14/7; elderly control subjects 5/64. The sensitivities and specificities were, respectively: 95 and 91% for PD versus elderly control; 97 and 91% for DLB versus elderly control; 88 and 91% for ADLS versus elderly control; 88 and 81% for ADLS versus ADNLS; 67 and 91% for ILBD versus elderly control. Olfactory bulb synucleinopathy density scores correlated significantly with synucleinopathy scores in all other brain regions (Spearman R values between 0.46 and 0.78) as well as with scores on the Mini-Mental State Examination and Part 3 of the Unified Parkinson’s Disease Rating Scale (Spearman R −0.27, 0.35, respectively). It is concluded that olfactory bulb LTS accurately predicts the presence of LTS in other brain regions. It is suggested that olfactory bulb biopsy be considered to confirm the diagnosis in PD subjects being assessed for surgical therapy.

Evaluation of α-synuclein immunohistochemical methods used by invited experts

The use of α-synuclein immunohistochemistry has altered our concepts of the cellular pathology, anatomical distribution and prevalence of Lewy body disorders. However, the diversity of methodology between laboratories has led to some inconsistencies in the literature. Adoption of uniformly sensitive methods may resolve some of these differences. Eight different immunohistochemical methods for demonstrating α-synuclein pathology, developed in eight separate expert laboratories, were evaluated for their sensitivity for neuronal elements affected by human Lewy body disorders. Identical test sets of formalin-fixed, paraffin-embedded sections from subjects diagnosed neuropathologically with or without Lewy body disorders were stained with the eight methods and graded by three observers for specific and nonspecific staining. The methods did not differ significantly in terms of Lewy body counts, but varied considerably in their ability to reveal neuropil elements such as fibers and dots. One method was clearly superior for revealing these neuropil elements and the critical factor contributing to its high sensitivity was considered to be its use of proteinase K as an epitope retrieval method. Some methods, however, achieved relatively high sensitivities with optimized formic acid protocols combined with a hydrolytic step. One method was developed that allows high sensitivity with commercially available reagents.

Neocortical Synapse Density and Braak Stage in The Lewy Body Variant of Alzheimer Disease: A Comparison with Classic Alzheimer Disease and Normal Aging.

Abstract. Substantial numbers of cortical and subcortical Lewy bodies are seen in approximately one quarter of patients whose brains show sufficient histopathologic changes for a neuropathologic diagnosis of definite Alzheimer disease (AD). This subset of cases has been named the Lewy body variant of AD (LBV). Despite comparable dementia and the presence of neocortical senile plaques in LBV patients, the overall burden of neuropathologic changes, in particular neurofibrillary tangles (NET), is less than in classic AD. While NFT frequency correlates with dementia severity in classic AD, the cognitive impairment in patients with LBV cannot be completely explained by such changes. Since several studies have suggested a role for synapse loss in relation to dementia severity in classic AD, we decided to investigate the role of synapse loss as a candidate for the cognitive impairment of LBV. The Braak staging method is based upon the distribution and severity of neurofibrillary changes, and one therefore would expect LBV cases to be assigned to lower Braak stages. In the present study we assigned a Braak stage to 14 LBV cases, 31 classic AD cases, and a group of 10 non-demented aged controls. We compared the severity of synapse loss as determined by ELISA immunoassay for synaptophysin and Braak stage among the three diagnostic groups. When compared to normal controls, synaptophysin concentrations were statistically significantly lower in both demented groups. There was comparable synapse loss in LBV and AD despite significantly lower Braak stages in the LBV cases. These results suggest a major role for loss of synapses as the substrate of cognitive impairment in LBV.

Neuropathologic evidence that the lewy body variant of Alzheimer disease represents coexistence of Alzheimer disease and idiopathic Parkinson disease

We undertook this study to investigate the neuropathologic relationships among Alzheimer disease (AD), idiopathic Parkinson disease (PD), and the Lewy body variant of AD (AD/LBV). We retrieved 30 autopsy cases in which Lewy bodies (LB) had been identified in the substantia nigra (SN) in routine hematoxylin-eosin-stained sections. Twenty-two of the cases had a primary clinical diagnosis of dementia and neuropathologic changes of AD; 12 of these demented patients also had clinical parkinsonism. Eight cases had clinical and neuropathologic evidence of PD with minimal or no AD neuropathology, though 6 had clinical dementia. Controls consisted of 6 cases of AD without SN LB by hematoxylin-eosin, and 5 neurologically normal aged controls. Paraffin sections of SN, superior temporal gyrus, and cingulate gyrus from each case were immunostained with rabbit anti-ubiquitin antiserum, randomized, and analyzed individually by light microscopy, and the density of LB-like profiles in each section were graded. None of 5 nondemented aged controls showed any neocortical LB, even though 2 had significant numbers of incidental SN LB by ubiquitin immunostaining. Of 6 AD cases without SN LB by hematoxylin-eosin, 3 had rare SN LB on ubiquitin stain, 1 of which showed rare neocortical Lewy-like profiles. Seven of 8 PD cases showed neocortical LB, including the 6 with dementia. Twenty-one of 22 AD cases with SN LB showed ubiquitin-immunoreactive Lewy-like bodies in the neocortex that were statistically significantly greater in number than in either pure PD or pure AD cases. The frequent occurrence of LB in the neocortex in PD alone suggests that AD/LBV likely represents mixed AD/PD. However, AD neuropathology may favor or promote the formation of neocortical LB in patients who go on to develop mixed AD/PD pathology.

Constitutive and regulated expression of the mouse Dinb (Polκ) gene encoding DNA polymerase kappa

A recently discovered group of novel polymerases are characterized by significantly reduced fidelity of DNA synthesis in vitro. This feature is consistent with the relaxed fidelity required for the replicative bypass of various types of base damage that frequently block high fidelity replicative polymerases. The present studies demonstrate that the specialized DNA polymerase kappa (polkappa) is uniquely and preferentially expressed in the adrenal cortex and testis of the mouse, as well as in a variety of other tissues. The adrenal cortex is the sole site of detectable expression of the Polkappa gene in mouse embryos. This adrenal expression pattern is consistent with a requirement for polkappa for the replicative bypass of DNA base damage generated during steroid biosynthesis. The expression pattern of polkappa in the testis is specific for particular stages of spermatogenesis and is distinct from the expression pattern of several other low fidelity DNA polymerases that are also expressed during spermatogenesis. The mouse (but not the human) Polkappa gene is primarily regulated by the p53 gene and is upregulated in response to exposure to various DNA-damaging agents in a p53-dependent manner.

Beta-amyloid precursor protein staining of nonaccidental central nervous system injury in pediatric autopsies.

Immunohistochemical staining for beta-amyloid precursor protein (betaAPP) is a well-established marker of traumatic axonal injury in adults. Recent studies have used similar techniques to evaluate nonaccidental central nervous system injury (NAI) in infants and young children. In this prospective study, we report the results of betaAPP immunohistochemistry on the brain and spinal cord in 28 pediatric cases of NAI. BetaAPP-immunoreactive axons were present in 27/28 cases. Vascular axonal injury (VAI) due to brain swelling and secondary vascular compromise was the most common pattern of betaAPP immunoreactivity and was detected in 22 of 28 cases. Traumatic axonal injury was detected in 19/28 cases, although only eight of these cases showed brainstem staining, thus fulfilling the criteria for the diagnosis of diffuse traumatic axonal injury (dTAI). TAI and VAI were both present in 16/28 cases. Isolated TAI and VAI occurred in three and five cases, respectively. All children with isolated VAI were <18 months of age. An additional finding highlighted by betaAPP immunostaining was a penumbra of axonal injury adjacent to focal lesions, such as lacerations. We conclude that betaAPP immunohistochemistry aids in documenting trauma in nonaccidental central nervous system injury in infants and young children and that VAI is a common finding.

Differences in ERK activation in squamous mucosa in patients who have gastroesophageal reflux disease with and without Barrett's esophagus.

OBJECTIVES:In some patients with gastroesophageal reflux disease (GERD), the reflux-damaged esophageal squamous epithelium heals through the process of intestinal metaplasia (resulting in Barretts esophagus) rather than through the regeneration of more squamous cells. We hypothesized that squamous epithelium in Barretts esophagus might have abnormalities in activation of the extracellular-regulated kinases 1 and 2 (ERK1/2) signaling pathway that may facilitate esophageal repair through metaplasia in response to acid-induced injury.METHODS:Endoscopic biopsies were taken from distal esophageal squamous mucosa in patients who had GERD with and without Barretts esophagus and in controls, before and after esophageal perfusion with 0.1 N HCl acid. Basal ERK1/2 phosphorylation, acid-induced ERK1/2 activity and phosphorylation, and localization of phosphorylated ERK1/2 were determined using immunoblotting, Western blotting, and immunohistochemistry.RESULTS:Compared to patients with Barretts esophagus, patients with GERD exhibited significantly lower baseline levels of phosphorylated ERK1/2 expression (35 ± 4% vs 90 ± 21% control, P = 0.01) Acid exposure significantly increased ERK1/2 activity (346.6 ± 51.90 to 446.8 ± 62.44 RIU, P = 0.02) and phosphorylation (3.55 ± 1.26 to 4.49 ± 1.25 [ratio phospho/total ERK], P = 0.01) in the squamous mucosa of GERD patients, but not in those with Barretts esophagus or in controls.CONCLUSIONS:Between patients with Barretts esophagus and patients with uncomplicated GERD, there are significant differences in baseline levels and in acid-induced activation of ERK1/2 in esophageal squamous epithelium. To our knowledge, this is the first description of a molecular, phenotypic feature that distinguishes the esophageal squamous mucosa of GERD patients with and without Barretts esophagus.

Neuroanatomic Profile of Polyglutamine Immunoreactivity in Huntington Disease Brains

A pathologic hallmark of Huntington disease (HD) is the presence of intraneuronal aggregates of polyglutamine-containing huntingtin protein fragments. Monoclonal antibody 1C2 is a commercial antibody to normal human TATA-binding protein that detects long stretches of glutamine residues. Using 1C2 as a surrogate marker formutant huntingtin protein, we immunostained 19 HD cases, 10 normal controls, and 10 cases of frontotemporal degeneration with ubiquitinated inclusions as diseased controls. In the HD cases, there was consistent 1C2 immunoreactivity in the neocortex, striatum, hippocampus, lateral geniculate body, basis pontis, medullary reticular formation, and cerebellar dentate nucleus. The normal and diseased controls demonstrated 1C2 immunoreactivity only in the substantia nigra, locus coeruleus, and pituitary gland. Staining of 5 HD cases and 5 normal controls revealed a less consistent and less diagnostically useful morphologic immunoreactivity profile. These results indicate that widespread 1C2 immunoreactivity is present in diverse central nervous system areas in HD, and that in the appropriate setting, 1C2 staining can be a useful tool in the postmortem diagnosis of HD when neuromelanin-containing neuronal populations are avoided.

Alpha-Synuclein Expression in the Developing Human Brain

Alpha (α)-synuclein is a presynaptic protein, abnormal expression of which has been associated with neurodegenerative and neoplastic diseases. It is abundant in the developing vertebrate central nervous system (CNS), but less is known about its developmental expression in the human CNS. Immunohistochemical expression of α-synuclein was studied in 39 fetal, perinatal, pediatric, and adolescent brains. Perikaryal expression of α-synuclein is observed as early as 11-wk gestation in the cortical plate. Several discrete neuronal groups in the hippocampus, basal ganglia, and brain stem express perikaryal α-synuclein by 20-wk gestation, persisting through the first few years of life. In the cerebellum, α-synuclein is present by 21-wk gestation and persists into adult life as a coarse granular neuropil reaction product in the internal granular layer, and as a diffuse neuropil “blush” in the molecular layer. The germinal matrix, glia, endothelial cells, external granular layer, Pukinje cells, and dentate neurons are consistently negative for α-synuclein. We conclude that α-synuclein is expressed very early in human gestation, and that its distribution and temporal sequence of expression varies in discrete neuronal groups. Perikaryal α-synuclein starts disappearing from the neuronal cytosol in early childhood, and only the neuropil retains immunoreactivity into adulthood. The reappearance of α-synuclein in the adult neuronal cytosol in certain disease processes may represent reemergence of cues from an earlier developmental stage as part of a stress response.