Comparisons with Brain-urea Concentrations in AD and in HD
Our group has previously reported severe and regionally-widespread increases in brain-tissue urea concentrations in cases with AD dementia (9), and with HD dementia (10, 11). In these studies, AD and HD brain-tissue urea values were reported from multiple regions compared between cases and matched controls (Table 4, Fig. 2; refs (8–10)).
Although there are some differences in the methodologies used in the two former studies compared with the current one (such as previous use of gas chromatography-mass spectrometry, and some differences in the regions studied; Fig. 2), case-control tissue-urea fold-changes can be compared between PDD, HD, and AD (Table 4) across several brain regions.
Cases and controls in the HD study were slightly younger than those in the current PDD cohort, at an average of around three years for controls and eight years for cases (10, 11). Moreover, PMD was significantly lower than in the current PDD cohort, at an average of ~ 11 hours in both cases and controls. Cause of death in the HD cohort was most commonly bronchopneumonia in cases and heart failure in controls, but data on comorbidities and other vascular pathology were unavailable. Brain tissue-urea concentrations showed a similar average fold-increase to that measured in PDD of 3.5 in HD cases compared to controls (Table 4, Fig. 2). As in PDD, the increase was observed in the SN, CB, HP, MTG, CG, SCX, and MCX, as well as in other regions not included in the current PDD study, such as the putamen, globus pallidus, middle frontal gyrus, and entorhinal cortex.
The AD cases whose brain-tissue urea levels we measured were similar in age to the current PDD cohort, with an average of ~ 70 years in both cases and controls (9). PMD was also significantly shorter in this cohort, at an average of 9 hours in controls and 7 hours in cases. Causes of death were varied in both cases and controls, most commonly being due to heart or lung complications. Comorbidity and vascular pathology metadata were not available. Although absolute tissue-urea concentrations cannot be directly compared to the previous studies in AD brains, there was a higher average case-control fold-change in AD than that observed here in PDD, the average being ~ 5.3-fold, but with increases as high as 6.5-fold in the HP (9) (Table 4). Tissue-urea elevations in this AD cohort were observed in the HP, MTG, SCX, MCX, CG, and CB, as well as in the entorhinal cortex (which was not included in the current study). The AD cohort was similar in age to the current PDD cohort.
Table 4
Urea fold-changes in PDD, AD, and HD
Region
|
Fold-Change in PDD
(this study)
|
Fold-Changes in AD
(Xu et al, 2016)
|
Fold-Changes in HD
(Patassini et al, 2015)
|
CB
|
3.7
|
4.9
|
3.6
|
MCX
|
4.1
|
5.0
|
3.4
|
PVC
|
4.3
|
4.9
|
3.4
|
HP
|
4.2
|
6.5
|
3.6
|
SN
|
3.9
|
-
|
3.5
|
MTG
|
4.3
|
4.7
|
3.4
|
MED
|
4.6
|
-
|
-
|
CG
|
5.5
|
5.3
|
3.5
|
PONS
|
3.9
|
-
|
-
|
PUT
|
-
|
-
|
3.7
|
GP
|
-
|
-
|
3.6
|
MFG
|
-
|
-
|
3.0
|
ENT
|
-
|
5.6
|
2.8
|
Overall
|
4.3
(3.9–4.6)
|
5.3
(4.8–5.8)
|
3.5
(3.2–3.6)
|
Comparisons of case-control fold-changes in human brain urea levels between PDD, AD and HD. Case-control differences were significant for every region in every disease. Overall shows mean overall fold-change with ±95% confidence intervals.
Additional studies have also confirmed urea increases in AD (18) and HD (12) brains, although one study reported decreases in the HD striatum and no change in the HD frontal lobe (19). Such discrepancies may be contributed to by differences in methods used or between cohorts, such as differences in PMD. However, we have previously observed that PMD does not affect urea levels for up to 72 hours in rat brains (20), although this may not necessarily also be the case in human brains.
Together, the observations in PDD, AD, and HD suggest a shared pathogenic mechanism in these three diseases, despite apparent differences in causative processes and symptomology. There appear to be regional differences between diseases with respect to tissue-urea elevations. For example, AD showed the highest increases in the severely-affected HP (6.5-fold), whereas in HD the putamen showed slightly higher-than-average elevations (3.7-fold). These regional differences may contribute to differences in severity and pathogenesis in the different conditions. Interestingly, the CG showed the highest urea increase in PDD (5.5-fold); the anterior CG is involved in emotive states and emotionally-coded memories. All other studied regions of the PDD brain showed urea increments of around 3.5- to 4.5-fold. However, there were no statistically significant differences between different PDD-control regions or between different PDD-case regions. Greater power (by increasing the sample size), would probably be required to determine whether the fold-elevation observed in the CG is significantly greater than that of other regions of the PDD brain. If so, it is possible that the prominence of both motor and cognitive dysfunction in PDD correlates with similar tissue urea elevations in areas involved in both cognition and motor control processes – a future investigation into regional tissue urea dysregulation in PD brains without dementia could help elucidate this and would probably be a logical next step.
Urea’s main metabolic function is to provide a route for the excretion of nitrogen-containing moieties derived from toxic nitrogen-containing compounds. In the urea cycle, ammonia is converted into urea, which is then excreted from the body via the urine. Urea is mainly produced in the liver and is carried to other parts of the body via the blood stream. However, it is slow to cross the blood-brain barrier (BBB) (21), leading to the question as to whether cerebral urea does in fact enter the brain through the BBB, or whether it is also produced in the brain. There is little evidence for the presence of intracerebral urea cycle activity, although there have been limited observations of partial activity in some studies. For example, ornithine transcarbamylase, an enzyme essential to the urea cycle was initially reported to be expressed only in AD brains (22), but has also been observed in healthy control brains in limited amounts (23, 24). However, studies attempting to investigate the levels of other urea cycle enzymes in HD sheep model brains for example were unable to identify either ornithine transcarbamylase or carbamoyl phosphate synthetase I in the striatum (12). Additionally, a more recent large-scale proteomic study of six regions of human AD and control brains was unable to identify the presence of either of these urea cycle enzymes at the protein level (25). Some other urea cycle components such as adenosine (9, 26, 27), citrulline (28), and ornithine (18, 28) have been reported in the AD brain, as well as the HD brain (11, 19) – however, as these metabolites are also involved in other metabolic pathways, this does not necessarily indicate urea cycle activity. For example, adenosine is a core component of several widespread co-enzymes such as adenosine triphosphate (ATP), diphosphate (ADP), and monophosphate (AMP) which are crucial to a wide variety of metabolic pathways including the ETC (29), TCA cycle (30), and purine metabolism (31). As such, it seems likely that cerebral urea may be formed by an alternative process and not the urea cycle itself.
Several urea cycle intermediates have also been reported to be dysregulated in PD serum, plasma, and CSF. Arginine has been reported by some investigations to be decreased in PD serum (32) and CSF (33), although other reports have observed no changes in either the serum (14), CSF (32, 34), or plasma (33). There is one report of increased citrulline in PD serum (35), although another investigation reported no change (14), and further reports indicated no change in the CSF (33, 34) or plasma (33, 36). Increases in PD serum ornithine have been reported (14), with varying reports in the CSF of decrease (33), increase (37), or no change (34). Moreover, no changes have been reported by several groups in studies of plasma ornithine in PD (33, 36, 37). None of these urea cycle components have been reported on in the PD/PDD brain itself, and the investigations of peripheral levels in the plasma, CSF, and serum have reported inconsistent results. As such, this entire pathway, and the possibility of partial or whole urea cycle activity in the brain, presents a novel area for future investigation of PDD.
Protein dysregulation and neuronal death may lead to greater protein breakdown, and so to increased urea production. Disruptions to the BBB, as observed in PDD (38), may result in defective urea clearance from the brain or increased entry of urea from the bloodstream via urea transporters. Urea transporters are responsible for regulating movement of urea by facilitating urea diffusion, and are expressed in astrocytes and the BBB as well as outside the brain. Urea transporter B has been shown to be upregulated in the HD CB, which may reflect attempts to clear elevated urea levels in the HD brain (12). Urea transporters have not yet been investigated in PD/PDD, but may show similar perturbations to those reported in HD.
Urea accumulation caused by kidney failure is known to be toxic to the brain, leading to a condition called uraemic encephalopathy (39). High urea levels lead to synaptic loss and inhibition of long-term potentiation via carbamylation of mTOR in a chronic kidney disease mouse model (40). Carbamylation is a post-translational modification involving the addition of isocyanate, usually derived from urea, to protein-bound amino-acid residues, causing alterations in the structure and function of the affected proteins. Increased carbamylation has been observed in aging humans (41), and as a result of chronic kidney disease (42) as well as in AD with cerebrovascular disease (43). It has been shown that tau, which is aggregated in AD and also to a lesser degree in PDD, can be carbamylated, resulting in increased amyloid formation and tau accumulation (44). Whether α-synuclein may also be carbamylated is unknown, but it does contain several potentially-susceptible amino-acid residues, which might serve as target sites for carbamylation.
High urea levels in chronic kidney disease and renal failure have also been linked to increased oxidative stress (45). Oxidative stress is a well-recognised feature of PD, AD, and HD (19, 46) and is linked to other pathogenic mechanisms including mitochondrial dysfunction and proteinopathy (47), dysregulated glucose metabolism (48), insulin resistance and inflammation (49), and α-synuclein accumulation, oligomerisation, and phosphorylation (50, 51). As such, it is possible that elevated urea levels in PDD could contribute to one or more of these known pathogenic mechanisms.