Variable expression of mitochondrial complex IV in the course of nigral intracellular accumulation of α-synuclein.

INTRODUCTION: Parkinson's disease (PD) is a neurodegenerative disease characterized by the deposition of disease-associated α-synuclein, which is thought to follow a sequential distribution in the human brain. Accordingly, α-Synuclein pathology affects the substantia nigra (SN) only in Braak stage 3 out of 6. Moreover, intracellular accumulation of α-synuclein follows maturation from non-ubiquitinated (p62 negative) to ubiquitinated (p62 positive) forms (Lewy bodies). Mitochondrial dysfunction is thought to be a central player in the pathogenesis of PD. It is not clear whether the nigral neurons already show mitochondrial alterations in stages preceding the deposition of α-synuclein in the SN, and how deposition of pre-aggregates or ubiquitinated mature inclusions relate to this. METHODS: Using cell-based morphometric immunohistochemistry we evaluated the volume density of mitochondrial complex-IV (COX-IV) immunoreactivity in SN neurons lacking or showing α-synuclein deposits in non-diseased individuals and those with Lewy body pathology Braak stage <3 lacking nigral α-synuclein pathology and Braak stage >3 with prominent nigral α-synuclein deposition. RESULTS: Increased volume density of COX-IV immunoreactivity appears before detectable pathological α-synuclein in nigral neurons. The volume density decreases significantly as pathological pre-aggregates of α-synuclein accumulates in the neurons and remains at a low level in neurons with p62 positive Lewy bodies. CONCLUSIONS: COX-IV expression shows a change before and during accumulation of α-synuclein in the SN underpinning the role of early mitochondrio protective therapy strategies in PD.

Early Selective Vulnerability of the CA2 Hippocampal Subfield in Primary Age-Related Tauopathy.

Primary age-related tauopathy (PART) is a neurodegenerative entity defined as Alzheimer-type neurofibrillary degeneration primarily affecting the medial temporal lobe with minimal to absent amyloid-ß (Aß) plaque deposition. The extent to which PART can be differentiated pathoanatomically from Alzheimer disease (AD) is unclear. Here, we examined the regional distribution of tau pathology in a large cohort of postmortem brains (n = 914). We found an early vulnerability of the CA2 subregion of the hippocampus to neurofibrillary degeneration in PART, and semiquantitative assessment of neurofibrillary degeneration in CA2 was significantly greater than in CA1 in PART. In contrast, subjects harboring intermediate-to-high AD neuropathologic change (ADNC) displayed relative sparing of CA2 until later stages of their disease course. In addition, the CA2/CA1 ratio of neurofibrillary degeneration in PART was significantly higher than in subjects with intermediate-to-high ADNC burden. Furthermore, the distribution of tau pathology in PART diverges from the Braak NFT staging system and Braak stage does not correlate with cognitive function in PART as it does in individuals with intermediate-to-high ADNC. These findings highlight the need for a better understanding of the contribution of PART to cognitive impairment and how neurofibrillary degeneration interacts with Aß pathology in AD and PART.

GPR55 controls functional differentiation of self-renewing epithelial progenitors for salivation.

GPR55, a lipid-sensing receptor, is implicated in cell cycle control, malignant cell mobilization, and tissue invasion in cancer. However, a physiological role for GPR55 is virtually unknown for any tissue type. Here, we localize GPR55 to self-renewing ductal epithelial cells and their terminally differentiated progeny in both human and mouse salivary glands. Moreover, we find GPR55 expression downregulated in salivary gland mucoepidermoid carcinomas and GPR55 reinstatement by antitumor irradiation, suggesting that GPR55 controls renegade proliferation. Indeed, GPR55 antagonism increases cell proliferation and function determination in quasiphysiological systems. In addition, Gpr55-/- mice present ~50% enlarged submandibular glands with many more granulated ducts, as well as disordered endoplasmic reticuli and with glycoprotein content. Next, we hypothesized that GPR55 could also modulate salivation and glycoprotein content by entraining differentiated excretory progeny. Accordingly, GPR55 activation facilitated glycoprotein release by itself, inducing low-amplitude Ca2+ oscillations, as well as enhancing acetylcholine-induced Ca2+ responses. Topical application of GPR55 agonists, which are ineffective in Gpr55-/- mice, into adult rodent submandibular glands increased salivation and saliva glycoprotein content. Overall, we propose that GPR55 signaling in epithelial cells ensures both the life-long renewal of ductal cells and the continuous availability of saliva and glycoproteins for oral health and food intake.

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 Parkinson's 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.

Novel approach for accurate tissue-based protein colocalization and proximity microscopy.

Fluorescence colocalization microscopy is frequently used to assess potential links between distinct molecules; however, this method can lead to striking false-positive results and erroneous conclusions. Here we developed a novel approach with more sophisticated mathematical colocalization analyses together with visualization of physical proximity using fluorescence resonance energy transfer (FRET). To verify our results we used the proximity ligation assay (PLA). With these methods we could demonstrate that distinct neurodegeneration-related proteins either not or only rarely interact in human brain tissue.

Secretagogin-dependent matrix metalloprotease-2 release from neurons regulates neuroblast migration.

The rostral migratory stream (RMS) is viewed as a glia-enriched conduit of forward-migrating neuroblasts in which chemorepulsive signals control the pace of forward migration. Here we demonstrate the existence of a scaffold of neurons that receive synaptic inputs within the rat, mouse, and human fetal RMS equivalents. These neurons express secretagogin, a Ca2+-sensor protein, to execute an annexin V-dependent externalization of matrix metalloprotease-2 (MMP-2) for reconfiguring the extracellular matrix locally. Mouse genetics combined with pharmacological probing in vivo and in vitro demonstrate that MMP-2 externalization occurs on demand and that its loss slows neuroblast migration. Loss of function is particularly remarkable upon injury to the olfactory bulb. Cumulatively, we identify a signaling cascade that provokes structural remodeling of the RMS through recruitment of MMP-2 by a previously unrecognized neuronal constituent. Given the life-long presence of secretagogin-containing neurons in human, this mechanism might be exploited for therapeutic benefit in rescue strategies.

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.

Shared and Distinct Patterns of Oligodendroglial Response in α-Synucleinopathies and Tauopathies.

Pathological protein deposits in oligodendroglia are common but variable features of various neurodegenerative conditions. To evaluate oligodendrocyte response in neurodegenerative diseases (NDDs) with different extents of oligodendroglial protein deposition we performed immunostaining for tubulin polymerization-promoting protein p25α (TPPP/p25α), α-synuclein (α-syn), phospho-tau, ubiquitin, myelin basic protein, and the microglial marker HLA-DR. We investigated cases of multiple system atrophy ([MSA] n = 10), Lewy body disease ([LBD] n = 10), globular glial tauopathy ([GGT] n = 7) and progressive supranuclear palsy ([PSP] n = 10). Loss of nuclear TPPP/p25α immunoreactivity correlated significantly with the degree of microglial reaction and loss of myelin basic prtein density as a marker of tract degeneration. This was more prominent in MSA and GGT, which, together with enlarged cytoplasmic TPPP/p25α immunoreactivity and inclusion burden allowed these disorders to be grouped as predominant oligodendroglial proteinopathies. However, distinct features, ie more colocalization of α-syn than tau with TPPP/p25α, more obvious loss of oligodendrocyte density in MSA, but more prominent association of tau protein inclusions in GGT to loss of nuclear TPPP/p25α immunoreactivity, were also recognized. In addition, we observed previously underappreciated oligodendroglial α-synuclein pathology in the pallidothalamic tract in LBD. Our study demonstrates common and distinct aspects of oligodendroglial involvement in the pathogenesis of diverse NDDs.

Familial early-onset dementia with complex neuropathologic phenotype and genomic background.

Despite significant progress in our understanding of hereditary neurodegenerative diseases, the list of genes associated with early-onset dementia is not yet complete. In the present study, we describe a familial neurodegenerative disorder characterized clinically as the behavioral and/or dysexecutive variant of Alzheimer's disease with neuroradiologic features of Alzheimer's disease, however, lacking amyloid-ß deposits in the brain. Instead, we observed a complex, 4 repeat predominant, tauopathy, together with a TAR DNA-binding protein of 43 kDa proteinopathy. Whole-exome sequencing on 2 affected siblings and 1 unaffected aunt uncovered a large number of candidate genes, including LRRK2 and SYNE2. In addition, DDI1, KRBA1, and TOR1A genes possessed novel stop-gain mutations only in the patients. Pathway, gene ontology, and network interaction analysis indicated the involvement of pathways related to neurodegeneration but revealed novel aspects also. This condition does not fit into any well-characterized category of neurodegenerative disorders. Exome sequencing did not disclose a single disease-specific gene mutation suggesting that a set of genes working together in different pathways may contribute to the etiology of the complex phenotype.

Dura mater is a potential source of Aß seeds.

Deposition of amyloid-ß (Aß) in the brain parenchyma and vessels is one of the hallmarks of Alzheimer disease (AD). Recent observations of Aß deposition in iatrogenic Creutzfeldt-Jakob disease (iCJD) after dural grafting or treatment with pituitary extracts raised concerns whether Aß is capable of transmitting disease as seen in prion diseases by the disease-associated prion protein. To address this issue, we re-sampled and re-evaluated archival material, including the grafted dura mater of two cases with iCJD (28 and 33-years-old) without mutations in the AßPP, PSEN1 and PSEN2 genes, and carrying ε3/ε3 alleles of the APOE gene. In addition, we evaluated 84 dura mater samples obtained at autopsy (mean age 84.9 ± 0.3) in the community-based VITA study for the presence of Aß deposition. We show that the dura mater may harbor Aß deposits (13 %) in the form of cerebral amyloid angiopathy or amorphous aggregates. In both iCJD cases, the grafted dura mater had accumulated Aß. The morphology and distribution pattern of cerebral Aß deposition together with the lack of tau pathology distinguishes the Aß proteinopathy in iCJD from AD, from that seen in young individuals without cognitive decline carrying one or two APOE4 alleles, and from that related to traumatic brain injury. Our novel findings of Aß deposits in the dura mater, including the grafted dura, and the distinct cerebral Aß distribution in iCJD support the seeding properties of Aß. However, in contrast to prion diseases, our study suggests that such Aß seeding is unable to reproduce the full clinicopathological phenotype of AD.

Amyloid-ß pathology and cerebral amyloid angiopathy are frequent in iatrogenic Creutzfeldt-Jakob disease after dural grafting.

QUESTIONS UNDER STUDY: Alzheimer-type amyloid-ß (Aß) pathology was reported in brains of individuals developing iatrogenic Creutzfeldt-Jakob disease (iCJD) after treatment with human cadaveric growth hormone, and interpreted as evidence of human transmission of Aß by the treatment. Here we investigated the prevalence of Aß pathology in other instances of iCJD related to dura mater grafts. METHODS: By use of immunohistochemistry for Aß, we investigated seven brains of patients (age range 28-63) who succumbed to iCJD after dural grafting, which had been applied by means of neurosurgery between 11 and 25 years before death. For control, we examined a series of 21 brains of age-matched (40-63 years) patients with sporadic CJD (sCJD) and an additional series of 81 sCJD cases (55-85 years) with the same methods. RESULTS: In five of seven iCJD brains, Aß was deposited in meningeal vessels as congophilic amyloid angiopathy and brain parenchymal plaques. This was significantly (p <0.001) more frequent than in the age-matched sCJD controls and in the usual sCJD series. CONCLUSIONS: We conclude that congophilic amyloid angiopathy and brain parenchymal Aß plaques are frequent in iCJD after dural grafting. The presence of Aß pathology in young individuals is highly unusual and suggests a causal relationship to the dural grafts. Further studies will be needed to elucidate whether such pathology resulted from the seeding of Aß aggregates from the grafts to host tissues.

Gaucher cells are not associated with α-synuclein neuropathology in infants.

A link between Gaucher disease (GD) and Parkinson disease (PD) has been suggested by many studies. Lewy-body-like α-synuclein inclusions have been shown in older GD patients who developed Parkinsonism. It has been proposed that decreased levels of glucocerebrosidase mediate impaired α-synuclein degradation and hence its accumulation. Nevertheless, this phenomenon is less investigated in the infantile form of GD. The aim of the study was to evaluate α-synuclein and τ-pathology in the brain and non-neural tissues (liver, spleen, pancreas, myocardial muscle, and lung) of 5 infants (age range from 1 month to 4 years) with GD. Our immunohistochemical study did not provide evidence that pathological α-synuclein or τ-accumulates early in tissues where Gaucher cells are already prominently present. Although recent finding of altered plasma α-synuclein levels in infantile GD patients suggest an early imbalance of α-synuclein homeostasis, our findings indicate that this does not inevitably coincide with α-synuclein pathology in the brain. Understanding this temporal variance of plasma levels and brain accumulation of α-synuclein might also be important when interpreting blood-based α-synuclein assays for the diagnosis of PD.

Heroin abuse exaggerates age-related deposition of hyperphosphorylated tau and p62-positive inclusions.

The observation of increased hyperphosphorylated tau levels correlating with microglial activation in opiate abusers has been interpreted as predisposition to accelerated Alzheimer disease-related changes. The present study focused on evaluating additional neurodegeneration-related proteins, including α-synuclein and TDP-43, and p62-positive deposits. We performed a systematic mapping of protein deposits in the brains of 27 individuals with documented heroin addiction (age: 19-40 years) and compared with 11 controls (age: 15-40 years). We confirm previous findings that heroin addiction associates with tau hyperphosphorylation in predilection brain areas for aging and Alzheimer disease. Furthermore, we show that this occurs also in areas implicated in the molecular disturbances and in vivo neuronal networks related to heroin abuse. There was, however, no presence of amyloid-beta deposits. We extend previous findings by showing the lack of TDP-43 or α-synuclein pathology and emphasize the independent effect of the duration of drug use on the appearance of age-related p62-positive neuritic profiles. These observations provide unique insights about neuropathological alterations in the brains of young heroin addicts and have implications about brain aging and the influences of environmental and toxic factors.

The role of proto-oncogene GLI1 in pituitary adenoma formation and cell survival regulation.

The Hedgehog (Hh) pathway is an important regulator of early tissue patterning and stem cell propagation. It was found to be aberrantly activated in numerous types of human cancer and might be relevant in cancer stem cells. The identification of adult stem cells in the pituitary raised the question if tumor-initiating cells and Hh signaling are involved in pituitary adenoma formation. The present study aimed at the evaluation of Hh signaling in relation to stem cell and cell cycle markers in 30 human pituitary adenomas and in cultured murine adenoma cells. Therefore, expression levels of components of the Hh pathway, stem cell marker SOX2, cell cycle regulator tumor-protein 53 (TP53), proliferation marker Ki67 (MKI67) and superoxide dismutase 1 (SOD1) were evaluated in 30 human pituitary adenomas in comparison to control tissue. Modulation of cell function and target gene expression by the inhibition and activation of the Hh pathway were studied in murine adenoma cells. We show that transcription factor glioma-associated oncogene 1 (GLI1) is overexpressed in 87% of all pituitary adenomas. The expression of GLI1 significantly correlated with that of SOX2, TP53, MKI67 and SOD1. Inhibition of GLI1 resulted in the downregulation of the above genes and severe cell death in mouse adenoma cells. On the other hand, activation of the Hh pathway increased cell viability and target gene expression. In conclusion, our findings point toward an alternative, ligand-independent Hh pathway activation with GLI1 playing a major role in the cell survival of pituitary adenoma cells.

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.