Ann E. Kingsbury

Tissue pH as an indicator of mRNA preservation in human post-mortem brain

The relationship between pH and mRNA preservation in post-mortem human brain was examined using in situ hybridization histochemistry and Northern hybridization with oligonucleotide probes in a large group of human subjects, including control and neuropathological cases. Tissue pH was found to correlate strongly with preservation of four mRNA species in three brain areas. Tissue with low pH, assumed to result from prolonged terminal hypoxia, contained reduced or absent mRNA, while tissue with higher pH was found to contain quantifiable amounts, the values for pathological brain samples being comparable to those for control material of similar pH. Measurement of tissue pH provides a simple means to screen post-mortem brain for mRNA preservation and is suggested as a means to match material in case-control studies of human neurodegenerative disease.

Brain stem pathology in Parkinson's disease: An evaluation of the Braak staging model

The lower brain stem of 25 pathologically‐confirmed Parkinsons disease (PD) cases was examined by alpha synuclein immunohistochemistry to characterize pathological accumulation of alpha synuclein (Lewy‐type α‐synucleinopathy, LTS) in the medulla oblongata, to examine differences between affected regions and test a proposed model of staging of pathology in PD. All cases had LTS in the medulla, including the dorsal motor nucleus of the vagus (dmX), when present. The distribution followed a consistent pattern and appeared to be concentrated in a tyrosine hydroxylase (TH) immunoreactive region, probably representing the dorsal IX/X nuclear complex and the intermediate reticular zone. LTS density was greatest in the dmX. A similar distribution pattern to PD was seen in 14 incidental Lewy body disease (ILBD) cases, five derived from the Queen Square Brain Bank tissue collection and nine identified in separate series of 60 neurologically‐normal individuals, and in three cases with the G2019S mutation of LRRK2. Semiquantitative assessment showed that severity of pathology in the dmX was not correlated with the severity of cortical pathology. Semiquantitative assay of TH and ChAT peptide expression in the medulla showed that TH expression in PD and ILBD did not differ from controls. These findings broadly support the Braak hypothesis of caudo‐rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one another.

Alteration in α-synuclein mRNA expression in Parkinson's disease

The presynaptic protein α‐synuclein is considered to play an important role in the pathophysiology of Parkinsons disease (PD). Point mutations in the α‐synuclein gene have been demonstrated in familial PD and α‐synuclein is a major component of Lewy bodies, the pathological hallmark of the sporadic disease. It is not clear whether abnormal accumulation of α‐synuclein is the result of abnormal levels of expression of the gene in neurodegenerative conditions. Expression of α‐synuclein mRNA was therefore studied in control and PD brain using semiquantitative in situ hybridization. α‐synuclein was expressed widely and hybridization signal was seen in most cortical regions, hippocampus, cerebellum, and brain stem. There was little mRNA in the striatum and no hybridization signal was detected in glia. High levels of α‐synuclein mRNA expression in neurons did not seem to be a marker for Lewy body formation. Abundant signal was seen both in regions in which Lewy body deposition occurs commonly in idiopathic PD (PD), such as substantia nigra and frontal and temporal cortex, as well as in less susceptible regions, e.g. visual cortex. Quantitative comparison of mRNA expression in regions of predilection for Lewy body formation showed that mRNA expression was reduced significantly in melanized substantia nigra neurons and frontal cortex neurons in Parkinsons disease. In substantia nigra neurons there seemed to be a negative correlation between cellular mRNA expression and disease duration. These findings are in broad agreement with other studies of the expression of α‐synuclein mRNA in human brain and suggest that Lewy body formation is unlikely to be the result of overexpression of α‐synuclein.

Regional differences in the severity of Lewy body pathology across the olfactory cortex.

We studied alpha-synuclein pathology in the rhinencephalon of ten cases of Parkinsons disease (PD) and twelve neurologically normal controls, of which seven had incidental Lewy bodies in the substantia nigra at autopsy and five had no pathological evidence of neurological disease. In all PD and incidental Lewy bodies cases, alpha-synuclein pathology was found in all five subregions of the primary olfactory cortex that were sampled, and amongst them the pathology was significantly more severe in the temporal division of the piriform cortex than in the frontal division of the piriform cortex, olfactory tubercle or anterior portions of the entorhinal cortex. The orbitofrontal cortex, which is an area of projection from the primary olfactory cortex, was affected in some cases but overall the alpha-synuclein pathology was less severe in this area than in the primary olfactory cortex. Because different areas of the rhinencephalon are likely to play different roles in olfaction and our data indicate a differential involvement by alpha-synuclein deposition of structures implicated in smell, future prospective studies investigating the pathophysiological basis of hyposmia in PD should consider to examine the areas of primary olfactory cortex separately.

Hyposmia in G2019S LRRK2-related parkinsonism Clinical and pathologic data

Background: Mutations in PARK8 (LRRK2) are associated with autosomal dominant parkinsonism and Parkinson disease (PD). Hyposmia is present in at least 80% of patients with PD and an accumulation of α-synuclein (α-syn) is seen in the olfactory pathways. In this study we have clinically examined olfaction and pathologically examined the rhinencephalon in individuals carrying the G2019S LRRK2 mutation. Methods: The University of Pennsylvania Smell Test (UPSIT) was used to evaluate the sense of smell in 19 parkinsonian and two asymptomatic carriers of the G2019S mutation and compared with groups of patients with PD and healthy controls. Postmortem examination of α-syn accumulation in the rhinencephalon was also carried out in four parkinsonian carriers of the G2019S mutation. Results: The mean UPSIT score in G2019S parkinsonian carriers was lower than that in healthy controls (p < 0.001) and similar to that found in patients with PD (p > 0.999). Smell tests in two asymptomatic carriers of the G2019S mutation were in the normal range. Postmortem studies of the olfactory pathways in one of the patients who had been clinically tested, and found to have hyposmia, and three other cases with the G2019S mutation, revealed α-syn deposition in the olfactory pathways in all cases. Conclusions: Odor identification is diminished in LRRK2 G2019S mutation parkinsonism but the asymptomatic carriers of the mutation had normal olfaction. We found α-syn accumulation with Lewy bodies in the rhinencephalon in all four cases examined pathologically.

Synphilin-1 and parkin show overlapping expression patterns in human brain and form aggresomes in response to proteasomal inhibition

Lewy bodies (LBs) are the characteristic inclusions of Parkinsons disease brain but the mechanism responsible for their formation is obscure. Lewy bodies (LBs) are composed of a number of proteins of which alpha-synuclein (alpha-SYN) is a major constituent. In this study, we have investigated the distribution patterns of synphilin-1 and parkin proteins in control and sporadic PD brain tissue by immunohistochemistry (IH), immunoblotting, and immunoelectron microscopy (IEM). We demonstrate the presence of synphilin-1 and parkin in the central core of a majority of LBs using IH and IEM. Using IH, we show an overlapping distribution profile of the two proteins in central neurons. Additionally, we show sensitivity of both endogenous synphilin-1 and parkin to proteolytic dysfunction and their co-localization in aggresomes formed in response to the proteasome inhibitor MG-132. We confirm that synphilin-1 and parkin are components of majority of LBs in Parkinsons disease and that both proteins are susceptible to proteasomal degradation.

Nitric oxide synthase mRNA expression in human subthalamic nucleus, striatum and globus pallidus: implications for basal ganglia function

The distribution of NOS mRNA within human basal ganglia was investigated using in situ hybridisation histochemistry (ISHH). Greater than 95% of subthalamic nucleus neurons were NOS mRNA-positive, between 1.5% and 2% of striatal neurons were positive and scattered NOS mRNA-positive neurons were detected in the medial, but not lateral globus pallidus. Levels of NOS mRNA expression per neuron were considerably higher in the striatum than in the pallidum or subthalamus. These findings have implications for basal ganglia function and disease states.

Hyposmia in progressive supranuclear palsy

Previous studies suggested that olfaction is normal in progressive supranuclear palsy (PSP). We applied the University of Pennsylvania Smell Identification Test (UPSIT) to 36 patients with PSP who scored more than 18 on the Mini Mental State Examination (MMSE), 140 patients with nondemented Parkinsons disease (PD) and 126 controls. Mean UPSIT scores in PSP were lower than in controls (P < 0.001) but higher than in PD (P < 0.001) after adjusting for age, gender, and smoking history. For patients with PSP, UPSIT scores correlated with MMSE (r = 0.44, P = 0.006) but not disease duration (P = 0.6), motor subscale of the Unified Parkinsons Disease Rating Scale (P = 0.2), or Frontal Assessment Battery (P = 0.5). The brains of six of the patients with PSP were examined postmortem and all revealed neurofibrillary tangles and tau accumulation in the rhinencephalon, although only three had hyposmia. Further prospective studies including patients with early PSP and PSP‐P with postmortem confirmation might help clarify if smell tests could be useful when the differential diagnosis lies between PD and PSP.

DJ-1 (PARK7) is associated with 3R and 4R tau neuronal and glial inclusions in neurodegenerative disorders.

Mutations in the DJ-1 gene are associated with autosomal recessive Parkinsons disease (PD), but its role in disease pathogenesis is unknown. This study examines DJ-1 immunoreactivity (DJ-1 IR) in a variety of neurodegenerative disorders, Alzheimers disease (AD), frontotemporal lobar degeneration (FTLD) with Pick bodies, FTLD with MAPT mutations, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), in which hyperphosphorylated tau inclusions are the major pathological signature. DJ-1 IR was seen in a subset of neurofibrillary tangles (NFTs), neuropil threads (NTs), and neurites in extracellular plaques in AD; tau inclusions in AD contained both 3R and 4R tau. A subset of Pick bodies in FTLD showed DJ-1 IR. In PSP, DJ-1 IR was present in a few NFTs, NTs and glial cell inclusions. In CBD, DJ-1 IR was seen only in astrocytic plaques. In cases of FTLD with MAPT mutations that were 4R tau positive (i.e. N279K and exon 10+16 mutations), DJ-1 IR was present mostly in oligodendroglial coiled bodies. However, in MAPT R406W mutation cases, DJ-1 IR was associated mainly with NFTs and NTs and these were both 3R and 4R tau positive. No DJ-1 IR was present in FTLD with ubiquitin inclusions (FTLD-U). In AD and FTLD with Pick bodies, DJ-1 protein was enriched in the sarkosyl-insoluble fractions of frozen brain tissue containing insoluble hyperphosphorylated tau, thus strengthening the association of DJ-1 with tau pathology. Additionally using two-dimensional gel electrophoresis, we demonstrated accumulation of acidic pI isoforms of DJ-1 in AD brain, which may compromise its normal function. Our observations confirm previous findings that DJ-1 is present in a subpopulation of glial and neuronal tau inclusions in tau diseases and associated with both 3R and 4R tau isoforms.

The corticotrophin-releasing factor-like peptide urocortin reverses key deficits in two rodent models of Parkinson's disease.

The potential neuroprotective action of the corticotrophin‐releasing factor‐related peptide urocortin (UCN) was investigated in the rat 6‐hydroxydopamine (6‐OHDA) and lipopolysaccharide (LPS) paradigms of Parkinsons disease. UCN (20 fmol) was either given at the same time as (T = 0) or 7 days after (T = +7) intracerebral 6‐OHDA or LPS injection. At 14 days after 6‐OHDA or LPS injection, circling behaviour was measured following apomorphine challenge. Circling was significantly lower in rats given UCN at either T = 0 or T = +7 compared with animals given 6‐OHDA or LPS and vehicle. Sham‐treated rats showed no circling. Consistent with these observations, striatal dopamine concentrations were markedly higher in 6‐OHDA/LPS + UCN rats vs. 6‐OHDA/LPS + vehicle groups. Additionally, l‐dihydroxyphenylalanine production by tyrosine hydroxylase was greatly reduced in the striata of 6‐OHDA/LPS + vehicle rats, whereas this was not the case in rats coadministered UCN. Finally, the numbers of tyrosine hydroxylase‐positive cells recorded in the substantia nigra of 6‐OHDA/LPS + vehicle‐treated animals were markedly lower than those of sham‐operated or 6‐OHDA/LPS + UCN rats. Critically, UCN was effective in reversing lesion‐induced deficits when given either at the same time as or 7 days after the neurotoxic insult. To our knowledge, this is the first time that such an effect has been demonstrated in vivo. The apparent ability of UCN to arrest the progression of or even reverse nigral lesions once established suggests that pharmacological manipulation of this system could have substantial therapeutic utility.