Günther Regelsberger

Distinct Patterns of Sirtuin Expression During Progression of Alzheimer’s Disease

Aging is one of the major risk factors for Alzheimer’s disease (AD). Sirtuins are associated with prolonged life span. To examine whether the expression levels of sirtuins associate with the progression of AD or not, we performed a comparative immunoblotting and immunohistochemical study of SIRT1, 3, and 5 in the entorhinal cortex and hippocampal subregions and white matter in 45 cases grouped according to Braak and Braak stages of neurofibrillary degeneration. In addition, we compared the expression levels with the local load of tau and amyloid-beta deposits, evaluated using morphometry. Our study revealed that (1) the neuronal subcellular redistribution of SIRT1 parallels the decrease in its expression, suggesting stepwise loss of neuroprotection dependent on the neuronal population; (2) in contrast to SIRT1 and 3, expression of SIRT5 increases during the progression of AD; (3) which might be related to its appearance in activated microglial cells. The complex patterns of the expression of sirtuins in relation to tissue damage should be taken into account when searching for therapies interacting with sirtuins.

X-linked adrenoleukodystrophy: very long-chain fatty acid metabolism is severely impaired in monocytes but not in lymphocytes

X-linked adrenoleukodystrophy (X-ALD) is a fatal neurodegenerative disease caused by mutations in the ABCD1 gene, encoding a member of the peroxisomal ABC transporter family. The ABCD1 protein transports CoA-activated very long-chain fatty acids (VLCFAs) into peroxisomes for degradation via β-oxidation. In the severest form, X-ALD patients suffer from inflammatory demyelination of the brain. As the extent of the metabolic defect in the main immune cells is unknown, we explored their phenotypes concerning mRNA expression pattern of the three peroxisomal ABC transporters, VLCFA accumulation and peroxisomal β-oxidation. In controls, ABCD1 expression was high in monocytes, intermediate in B cells and low in T cells; ABCD2 expression was extremely low in monocytes, intermediate in B cells and highest in T cells; ABCD3 mRNA was equally distributed. In X-ALD patients, the expression patterns remained unaltered; accordingly, monocytes, which lack compensatory VLCFA transport by ABCD2, displayed the severest biochemical phenotype with a 6-fold accumulation of C26:0 and a striking 70% reduction in peroxisomal β-oxidation activity. In contrast, VLCFA metabolism was close to control values in B cells and T cells, supporting the hypothesis that sufficient ABCD2 is present to compensate for ABCD1 deficiency. Thus, the vulnerability of the main immune cell types is highly variable in X-ALD. Based on these results, we propose that in X-ALD the halt of inflammation after allogeneic hematopoietic stem cell transplantation relies particularly on the replacement of the monocyte lineage. Additionally, these findings support the concept that ABCD2 is a target for pharmacological induction as an alternative therapeutic strategy.

Tau Pathology in Creutzfeldt-Jakob Disease Revisited.

Creutzfeldt‐Jakob disease (CJD) is a human prion disease with different etiologies. To determine the spectrum of tau pathologies in CJD, we assessed phospho‐Tau (pTau) immunoreactivities in 75 sporadic CJD cases including an evaluation of the entorhinal cortex and six hippocampal subregions. Twelve cases (16%) showed only small tau‐immunoreactive neuritic profiles. Fifty‐two (69.3%) showed additional tau pathology in the medial temporal lobe compatible with primary age related tauopathy (PART). In 22/52 cases the lower pTau immunoreactivity load in the entorhinal cortex as compared to subiculum, dentate gyrus or CA4 region of the hippocampus was significantly different from the typical distribution of the Braak staging. A further 11 cases (14.7%) showed widespread tau pathologies compatible with features of primary tauopathies or the gray matter type of ageing‐related tau astrogliopathy (ARTAG). Prominent gray matter ARTAG was also observed in two out of three additionally examined V203I genetic CJD cases. Analysis of cerebrospinal fluid revealed prominent increase of total tau protein in cases with widespread tau pathology, while pTau (T181) level was increased only in four. This correlated with immunohistochemical observations showing less pathology with anti‐pTau T181 antibody when compared to anti‐pTau S202/T205, T212/S214 and T231. The frequency of tau pathologies is not unusually high in sporadic CJD and does not precisely relate to PrP deposition. However, the pattern of hippocampal tau pathology often deviates from the stages of Braak. Currently applied examination of cerebrospinal fluid pTau (T181) level does not reliably reflect primary tauopathies, PART and ARTAG seen in CJD brains.

Peroxisomal Localization of the Proopiomelanocortin-Derived Peptides β-Lipotropin and β-Endorphin

The peptide hormones ACTH, MSHs, β-lipotropin (β-LPH), and β-endorphin are all derived from the precursor molecule proopiomelanocortin (POMC). Using confocal laser microscopy and immunoelectron microscopy in human pituitary gland, we demonstrate a peroxisomal localization of β-endorphin and β-LPH in cells expressing the peroxisomal ATP-binding cassette-transporter adrenoleukodystrophy protein (ALDP). The peroxisomal localization of β-LPH and β-endorphin was not restricted to the pituitary gland but was additionally found in other human tissues that express high levels of ALDP, such as dorsal root ganglia, adrenal cortex, distal tubules of kidney, and skin. In contrast to the peptide hormones β-LPH and β-endorphin, which are derived from the C terminus of POMC, the N-terminal peptides ACTH, α-MSH, and γ-MSH were never detected in peroxisomes. This novel peroxisomal localization of β-endorphin and β-LPH in ALDP-positive cells was confirmed by costaining with ALDP and the peroxisomal marker catalase. Moreover, peroxisomal sorting of β-LPH could be modeled in HeLa cells by ectopic expression of a POMC variant, modified to allow cleavage and release of β-LPH within the secretory pathway. Although β-LPH and β-endorphin were only associated with peroxisomes in cells that normally express ALDP, the transporter activity of ALDP is not necessary for the peroxisomal localization, as demonstrated in tissues of X-linked adrenoleukodystrophy patients lacking functional ALDP. It remains to be elucidated whether and how the peroxisomal localization of POMC-derived hormones has a role in the endocrine dysfunction of peroxisomal disease.

Cerebrotendinous xanthomatosis with the c.379C>T (p.R127W) mutation in the CYP27A1 gene associated with premature age-associated limbic tauopathy

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive disease associated with mutations in the mitochondrial CYP27A1 gene [1]. The deficiency of sterol 27-hydroxylase leads to the disturbance of bile acid synthesis and causes a multisystem disorder, including signs of premature ageing like cataracts or atherosclerosis. In addition, neuropsychiatric symptoms, like dementia, cerebellar ataxia, pyramidal signs, seizures or extrapyramidal symptoms may occur [1]. Magnetic resonance imaging (MRI) examinations emphasize cerebellar atrophy and bilateral lesions in the cerebellar white matter, while occasional neuropathological studies described granulomatous and xanthomatous changes mainly in the cerebellar hemispheres, globus pallidus and cerebellar peduncles [1]. There is a paucity of information whether age-related neurodegenerative diseases are present in CTX or not. We report the clinical, biochemical and neuropathological features of CTX associated with neuron-predominant tau pathology predominating in the limbic system, which was compatible with age-dependent argyrophilic grain disease. Two Hungarian adult siblings were examined. The elder brother died at the age of 46, and his brother is still alive at the age of 43 and presented with a similar clinical course (summarized in Table 1). The analyses presented here were performed as diagnostic tests and consent for publication was obtained from their mother. In both patients mitochondrial encephalopathy was suspected clinically. During the childhood of patient 1, diarrhoea and anaemia was reported. At elementary school a psychological examination showed moderate mental deficiency. At the age of 14 his IQ was 73. Ten years later hepatomegaly was noted. Two years later cataract was diagnosed in both eyes. Furthermore, due to cognitive impairment and aggression he left his job as an unskilled worker. Soon gait disorder and ataxia evolved and progressed to a spastico-paretico-ataxic gait, together with dysarthria, tetrapyramidal symptoms and tetraparesis. At the age of 45 he had epileptic seizures, his state deteriorated and he died due to bronchopneumonia. General autopsy showed severe atherosclerosis, fibrotic degeneration of myocardium and fatty degeneration of the liver. Neuropathological studies were performed as described using a panel of antibodies against neurodegeneration-related proteins [2], including amyloid-beta (A-beta; 1:50, clone 6F/3D, Dako, Glostrup, Denmark), alpha-synuclein (clone 5G4, 1:2000, Roboscreen, Leipzig, Germany), phospho-TDP-43 (pS409/ 410, 1:2000, Cosmo Bio, Tokyo, Japan), anti-p62 (1:500, BD Biosciences, San Jose, CA, USA), anti-ubiquitin (1:50 000, Millipore, Temecula, CA, USA), and a panel of anti-tau antibodies: anti-tau AT8 (pS202, 1:200), AT100 (pS212, 1:200), AT180 (pT231, 1:200), AT270 (pT181, 1:200), HT7 (tau 169-163, 1:100; all from Pierce Biotechnology, Rockford, IL, USA), anti-4R tau (RD4, 1:200, Upstate, Charlottesville, VA, USA), anti-3R tau (RD3, 1:2000, Upstate). The DAKO EnVision© detection kit, peroxidase/DAB, rabbit/mouse (Dako) was used for visualization of antibody reactions. In addition, Haematoxylin and Eosin, Luxol Fast Blue/nuclear fast red, periodic acidSchiff (PAS), Sudan-black, and Bielschowsky and Gallyas silver stainings were performed. Four major alterations were observed: (1) xanthomatous changes; (2) loss of axons and myelin in the white matter; (3) neuronal loss and reactive astrogliosis; and (4) limbic tauopathy compatible with argyrophilic grain disease. Accumulation of perivascular macrophages, multinucleated giant cells, fibrotic vessels and cholesterol clefts (Figure 1A–D) predominated in the cerebellar white matter, dentate nucleus, globus pallidus, cerebral peduncles, pontine base, and pyramids (Table 2). PAS positive granular material accumulated in the perivascular space; ultrastructurally these were composed of convolutes of membranes that were about 12–15 nm thick (Figure 1E,F). In addition these showed autofluorescence and were Sudan black positive indicative of lipids (online Figure S1). Prominent loss of myelin and axons (Figure 1G), and PAS-positive degradation products in macrophages together with perivascular CD8-positive T cells (Figure 1H) were seen mainly in the cerebellum, cerebral peduncles, pontine base (transverse fibres preserved) and pyramids. Neuronal loss was prominent in the inferior olives, dentate nucleus and cerebellar cortex associated with reactive microgliosis (Figure 1I). Reactive astrogliosis

Refsum disease due to the splice-site mutation c.135-2A>G before exon 3 of the PHYH gene, diagnosed eight years after detection of retinitis pigmentosa

OBJECTIVES If Refsum disease (RD) is not considered as a differential at onset of the initial manifestations the diagnosis of RD remains unrecognized for a long time as in the following case. CASE REPORT A 55-y old Caucasian female with hyperextensible joints developed progressive visual impairment due to retinitis pigmentosa and sensorimotor polyneuropathy of the lower limbs since age 32 y. Screening for causes of polyneuropathy at age 40 y revealed markedly elevated serum phytanic acid (PA) with a maximum value of 293.6 microg/ml (n:<6 microg/ml) why RD was diagnosed. Since age 48 y slowly progressive hypacusis was noted. RD was caused by the known transition A135G in exon 3 of the PHYH gene. Additionally, the polymorphism T153C in exon 3 of the PHYH gene was detected. Upon strict adherence to the Chelsea diet PA levels slightly decreased since onset of this therapy. CONCLUSION This case confirms that RD remains unrecognized for a long time if RD is not considered as a differential of retinitis pigmentosa as the initial manifestation of the disease. Early recognition of RD is important since there is the therapeutic option of starting a diet.

Plasma and cerebrospinal fluid tau and neurofilament concentrations in rapidly progressive neurological syndromes: a neuropathology-based cohort

Cerebrospinal fluid (CSF) tau and neurofilament light chain (NF‐L) proteins have proved to be reliable biomarkers for neuronal damage; however, there is a strong need for blood‐based tests.

Lysosomal response in relation to α-synuclein pathology differs between Parkinson's disease and multiple system atrophy

Intracellular deposition of pathologically altered α-synuclein mostly in neurons characterises Parkinsons disease (PD), while its accumulation predominantly in oligodendrocytes is a feature of multiple system atrophy (MSA). Recently a prion-like spreading of pathologic α-synuclein has been suggested to play a role in the pathogenesis of PD and MSA. This implicates a role of protein processing systems, including lysosomes, supported also by genetic studies in PD. However, particularly for MSA, the mechanism of cell-to-cell propagation of α-synuclein is yet not fully understood. To evaluate the significance of lysosomal response, we systematically compared differently affected neuronal populations in PD, MSA, and non-diseased brains using morphometric immunohistochemistry (cathepsin D), double immunolabelling (cathepsin D/α-synuclein) laser confocal microscopy, and immunogold electron microscopy for the disease associated α-synuclein. We found that i) irrespective of the presence of neuronal inclusions, the volume density of cathepsin D immunoreactivity significantly increases in affected neurons of the pontine base in MSA brains; ii) volume density of cathepsin D immunoreactivity increases in nigral neurons in PD without inclusions and with non-ubiquitinated pre-aggregates of α-synuclein, but not in neurons with Lewy bodies; iii) cathepsin D immunoreactivity frequently colocalises with α-synuclein pre-aggregates in nigral neurons in PD; iv) ultrastructural observations confirm disease-associated α-synuclein in neuronal and astrocytic lysosomes in PD; v) lysosome-associated α-synuclein is observed in astroglia and rarely in oligodendroglia and in neurons in MSA. Our observations support a crucial role for the neuronal endosomal-lysosomal system in the processing of α-synuclein in PD. We suggest a distinct contribution of lysosomes to the pathogenesis of MSA, including the possibility of oligodendroglial and eventually neuronal uptake of exogenous α-synuclein in MSA.

Impaired plasticity of macrophages in X-linked adrenoleukodystrophy

Weinhofer et al. reveal impaired plasticity of macrophages, with intrinsic pro-inflammatory skewing and a decreased ability to establish proper anti-inflammatory responses, in X-linked adrenoleukodystrophy (ALD). This may contribute to the rapidly progressive demyelination seen in cerebral ALD.

Non-manifesting Refsum heterozygotes carrying the c.135-2A>G PAHX gene transition

So far, subjects heterozygous for PAHX mutations are regarded as non-symptomatic. In the 24-year-old, HIV-negative daughter and the 26-year-old, HIV-negative son of a patient with Refsum disease due to the homozygous c.135-2A>G transition at the splice site before exon 3 of the PAHX gene, slight abnormalities suggestive of the disease became apparent. The daughter reported a single fever cramp in childhood, recurrent, short-lived amaurotic episodes after getting up from supine, short-sightedness, hypoacusis, and restless legs. The son complained about restless legs, hyperhidrosis, hypoacusis, and bulbar oscillations. Though both children carried the same mutation as their mother in the heterozygous form, clinical neurologic examination, nerve conduction studies and serum phytanic acid concentration were normal in both of them, implying that the described abnormalities were not causally related to the PAHX mutation. In the absence of elevated phytanic acid concentrations, clinical neurologic abnormalities in heterozygous relatives of Refsum patients are not attributable to heterozygosity for PAHX mutations.