Ultrastructural localization of Porphyromonas gingivalis gingipains in the substantia nigra of Parkinson's disease brains.

Gingipains are protease virulence factors produced by Porphyromonas gingivalis, a Gram-negative bacterium best known for its role in chronic periodontitis. Gingipains were recently identified in the middle temporal gyrus of postmortem Alzheimer's disease (AD) brains, where gingipain load correlated with AD diagnosis and tau and ubiquitin pathology. Since AD and Parkinson's disease (PD) share some overlapping pathologic features, including nigral pathology and Lewy bodies, the current study explored whether gingipains are present in the substantia nigra pars compacta of PD brains. In immunohistochemical techniques and multi-channel fluorescence studies, gingipain antigens were abundant in dopaminergic neurons in the substantia nigra of both PD and neurologically normal control brains. 3-dimensional reconstructions of Lewy body containing neurons revealed that gingipains associated with the periphery of alpha-synuclein aggregates but were occasionally observed inside aggregates. In vitro proteomic analysis demonstrated that recombinant alpha-synuclein is cleaved by lysine-gingipain, generating multiple alpha-synuclein fragments including the non-amyloid component fragments. Immunogold electron microscopy with co-labeling of gingipains and alpha-synuclein confirmed the occasional colocalization of gingipains with phosphorylated (pSER129) alpha-synuclein. In dopaminergic neurons, gingipains localized to the perinuclear cytoplasm, neuromelanin, mitochondria, and nucleus. These data suggest that gingipains localize in dopaminergic neurons in the substantia nigra and interact with alpha-synuclein.

Human pericytes degrade diverse α-synuclein aggregates.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder characterised by the abnormal accumulation of α-synuclein (α-syn) aggregates. Central to disease progression is the gradual spread of pathological α-syn. α-syn aggregation is closely linked to progressive neuron loss. As such, clearance of α-syn aggregates may slow the progression of PD and lead to less severe symptoms. Evidence is increasing that non-neuronal cells play a role in PD and other synucleinopathies such as Lewy body dementia and multiple system atrophy. Our previous work has shown that pericytes-vascular mural cells that regulate the blood-brain barrier-contain α-syn aggregates in human PD brains. Here, we demonstrate that pericytes efficiently internalise fibrillar α-syn irrespective of being in a monoculture or mixed neuronal cell culture. Pericytes cleave fibrillar α-syn aggregates (Fibrils, Ribbons, fibrils65, fibrils91 and fibrils110), with cleaved α-syn remaining present for up to 21 days. The number of α-syn aggregates/cell and average aggregate size depends on the type of strain, but differences disappear within 5 five hours of treatment. Our results highlight the role brain vasculature may play in reducing α-syn aggregate burden in PD.

Odorant receptor proteins in the mouse main olfactory epithelium and olfactory bulb.

In the mouse, odorant receptor proteins (ORs) are G-protein-coupled receptors expressed in mature olfactory sensory neurons (OSNs) of the main olfactory epithelium (MOE). ORs mediate odorant reception at the level of the OSN cilia. Most of the ∼1100 OR genes in the mouse genome are expressed, at the RNA level, in mature OSNs. The literature on antibodies against ORs is limited, and most reports are with antibodies that are not commercially available. Here we have screened 40 commercial antibodies against human and mouse ORs by immunofluorescence staining of coronal cryosections of the MOE of 21-day-old C57BL/6J mice. Various methods of antigen retrieval were tested. Of the 19 antibodies raised against human ORs, three yielded a consistent immunoreactive signal in the mouse MOE; of these three, two appeared to cross react against one or more, unknown, mouse ORs. Of the 21 antibodies raised against mouse ORs, six yielded a consistent immunoreactive signal in the mouse MOE; of these six, two also stained specific glomeruli in the olfactory bulb. Antibody specificity could be validated with gene-targeted mouse strains in the case of three ORs. The number of OSNs immunoreactive for the MOR28/Olfr1507 antibody is greater in C57BL/6J than in 129S6/SvEvTac wild-type mice. Taken together, our results are encouraging: 20-30% of these commercially available antibodies are informative in immunohistochemical analyses of the mouse MOE. The commercial availability of these antibodies should facilitate the study of OR proteins in the MOE and the olfactory bulb, and the replicability of results in the literature.

Distribution of PSA-NCAM in normal, Alzheimer's and Parkinson's disease human brain.

Polysialated neural cell adhesion molecule (PSA-NCAM) is a membrane bound glycoprotein widely expressed during nervous system development. While commonly described in the neurogenic niches of the adult human brain, there is limited evidence of its distribution in other brain regions. PSA-NCAM is an important regulator of cell-cell interactions and facilitates cell migration and plasticity. Recent evidence suggests these functions may be altered in neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's disease (PD). This study provides a detailed description of the PSA-NCAM distribution throughout the human brain and quantitatively compares the staining load in cortical regions and sub-cortical structures between the control, AD and PD brain. Our results provide evidence of widespread, yet specific, PSA-NCAM expression throughout the human brain including regions devoid of PSA-NCAM in the rodent brain such as the caudate nucleus (CN) and cerebellum (CB). We also detected a significant reduction in PSA-NCAM load in the entorhinal cortex (EC) of cases that was inversely correlated with hyperphosphorylated tau load. These results demonstrate that PSA-NCAM-mediated structural plasticity may not be limited to neurogenic niches and is conserved in the aged brain. We also provide evidence that PSA-NCAM is reduced in the EC, a region severely affected by AD pathology.

Netrin-1 stimulates developing GnRH neurons to extend neurites to the median eminence in a calcium- dependent manner.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are required for fertility in all mammalian species studied to date. In rodents, GnRH neuron cell bodies reside in the rostral hypothalamus, and most extend a single long neuronal process in the caudal direction to terminate at the median eminence (ME), the site of hormone secretion. The molecular cues that GnRH neurites use to grow and navigate to the ME during development, however, remain poorly described. Reverse transcription-PCR (RT-PCR) identified mRNAs encoding Netrin-1, and its receptor, DCC, in the fetal preoptic area (POA) and mediobasal hypothalamus (MBH), respectively, from gestational day 12.5 (GD12.5), a time when the first GnRH neurites extend toward the MBH. Moreover, a subpopulation of GnRH neurons from GD14.5 through GD18.5 express the Netrin-1 receptor, DCC, suggesting a role for Netrin-1/DCC signaling in GnRH neurite growth and/or guidance. In support of this notion, when GD15.5 POA explants, containing GnRH neurons actively extending neurites, were grown in three-dimensional collagen gels and challenged with exogenous Netrin-1 (100 ng/ml or 400 ng/ml) GnRH neurite growth was stimulated. In addition, Netrin-1 provided from a fixed source was able to stimulate outgrowth, although it did not appear to chemoattract GnRH neurites. Finally, the effects of Netrin-1 on the outgrowth of GnRH neurites could be inhibited by blocking either L-type voltage-gated calcium channels (VGCCs) with nifedipine (10 µM), or ryanodine receptors with ryanodine (10 µM). This is consistent with the role of Ca2+ from extra- and intracellular sources in Netrin-1/DCC-dependent growth cone motility in other neurons. These results indicate that Netrin-1 directly stimulates the growth of a subpopulation of GnRH neurites that express DCC, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional neuronal population whose neurites utilize Netrin-1/DCC signaling for their development.

Neurogenesis and progenitor cells in the adult human brain: a comparison between hippocampal and subventricular progenitor proliferation.

For more than a decade, we have known that the human brain harbors progenitor cells capable of becoming mature neurons in the adult human brain. Since the original landmark article by Eriksson et al. in 1998 (Nat Med 4:1313-1317), there have been many studies investigating the effect that depression, epilepsy, Alzheimer's disease, Huntington's disease, and Parkinson's disease have on the germinal zones in the adult human brain. Of particular interest is the demonstration that there are far fewer progenitor cells in the hippocampal subgranular zone (SGZ) compared with the subventricular zone (SVZ) in the human brain. Furthermore, the quantity of progenitor cell proliferation in human neurodegenerative diseases differs from that of animal models of neurodegenerative diseases; there is minimal progenitor proliferation in the SGZ and extensive proliferation in the SVZ in the human. In this review, we will present the data from a range of human and rodent studies from which we can compare the amount of proliferation of cells in the SVZ and SGZ in different neurodegenerative diseases.

Detection of small-fiber neuropathy by sudomotor testing.

The symptoms of burning sensation affecting the feet, thought to be due to a distal small-fiber neuropathy (DSFN) affecting somatic unmyelinated fibers, are usually accompanied by vasomotor or sudomotor changes suggestive of involvement of autonomic fibers. We therefore examined the relationship between pattern of anhidrosis and DSFN and its etiology, comparing patients with "pure" DSFN (with normal nerve conduction) to those with clinical DSFN (minor conduction abnormalities). We reviewed 125 cases with a clinical phenotype of DSFN. These patients had distal burning discomfort, variable sensory deficits, and intact motor function. All had undergone assessment with thermoregulatory sweat test (TST), autonomic reflex screen (ARS), and nerve conduction studies and electromyography (NCS/EMG). TST showed a distal pattern of anhidrosis in 74%. The quantitative sudomotor axon reflex test (QSART) was abnormal in 74%, with 80% of those having a length-dependent pattern of anhidrosis/hypohidrosis. In total, 93% of patients had a distal pattern of abnormality on QSART or TST. The Composite Autonomic Severity Score (CASS) was used to quantify the severity and distribution of autonomic deficits: 98% had CASS abnormality (sudomotor, 98%; adrenergic, 43%; cardiovagal, 35%). EMG was normal or showed unrelated abnormalities in 75%. The most common etiologies of DSFN were idiopathic (73%), presumed hereditary (18%), and diabetes (10%). Sudomotor examination is thus a highly sensitive detection tool in DSFN. Autonomic involvement is mainly distal, and additionally may involve adrenergic and the long cardiovagal fibers.