**6. Metabolic dysfunction and its link to Alzheimer's disease: The role of dehydrogenases**

Alzheimer's disease is the most common form of dementia characterized by loss of memory, cognitive decline and change in perception and behavior. Pathological hallmarks include accumulation of amyloid beta protein (Aβ) and resulting plaque formation (a cleavage protein of amyloid precursor protein-APP) and formation of neurofibrillary tangles (due to hyper phosphorylation of microtubule associated protein of neurons in the brain). Genetic mutations in APP protein or ApoE protein (a protein linked to lipid and cholesterol in the body) as well as Down's syndrome are some examples of genetic predispositions that increase the propensity to develop Alzheimer's disease. However, the etiology of the disease is still questionable. Both vascular as well as metabolic dysfunction have been accredited as major factors prodromic to the pathogenesis and progression of Alzheimer's disease (84). Vascular dysfunction includes reduction in cerebral blood flow, reduced glucose uptake, and reduced amyloid beta clearance with cerebral amyloid angiopathy. The patients with atherosclerosis have shown to have an increased risk to develop AD. Vascular structural anomalies in cerebral vessels (like increased pinocytic activity as well as swelling) have been seen in early AD further signifying the importance of vascular dysfunction in AD pathogenesis. Defective glucose utilization has been observed earlier than reduced cerebral blood flow (CBF), indicating the role of glucose metabolism in AD development. What is of primary interest to us is that it is slowly becoming well accepted that metabolic dysfunction, related oxidative stress and mitochondrial deficits can precede AD development (84-86). In one such recent study, triple transgenic mice 3xTg-AD were developed (having mutations in human APPSWE, TauP301L, PS1M146V genes linked with AD) along with their respective controls. Decreased mitochondrial respiration was observed along with reduced PDH activity. High levels of oxidative stress (via measurement of hydrogen peroxide production and related lipid peroxidation), high Aβ levels with high levels of Aβ binding alcohol dehydrogenase (ABAD) was observed. Decreased respiration was also observed in embryonic neurons, which continued till senescence leading to AD pathogenesis. Thus these studies clearly emphasized how mitochondrial dysfunction and resulting metabolic respiration preceded AD development. Defective enzyme function(of dehydrogenases) in pathways of energy metabolism such as glycolysis, tricarboxylic acid pathway and electron transport chain have been well studied in AD progression. Defective PDH, KGDHC, cytochrome oxidase along with reduced activity of hexokinase, phosphofructokinase are the major enzymes reported in AD so far (87). In the paragraph below we will be further discuss some of these DHOs and their mechanism of metabolic dysfunction in AD. We will also look at other DHOs apart from those directly involved in the bioenergetics which have an important role in pathogenesis of AD.

Role and Function of Dehydrogenases in CNS and Blood-Brain Barrier Pathophysiology 105

studies, it was hypothesized that ABAD can act as a crucial link between increased Aβ production and mitochondrial dysfunction in Alzheimer's disease progression (90-92).To test this hypothesis, double transgenic mice with increased levels of ABAD and Aβ were developed (along with Tg mAP, Tg ABAD, and not Tg littermate controls)(93). Neuron cultures derived from these Tg mice showed increased ROS, oxidative stress and relative decrease in ATP production. Further studies indicated defective activity of mitochondrial Complex IV as the source of the ROS species, also such effect was not observed in single Tg mice with increased ABAD alone (suggesting Aβ acting as a crucial element linking the two). Lactate dehydrogenase (LDH) was higher in the Tg mAP/ABAD mice as compared to other groups suggesting the reversal to lactate metabolism. Cell apoptosis via caspase 3 activity was observed in *in vitro* studies. Data from the Tg mice also suggested reduced ATP production at 9 months of age along with reduced Complex IV activity. Overall, ABAD acts a crucial enzyme that can lead to mitochondrial dysfunction

**Figure 6.** : Correlation between aging free radicals, DHOs and the onset of CNS disorders.

and disease progression in AD.

One such DHO as mentioned earlier is Aβ binding alcohol dehydrogenase (ABAD). ABAD is a short-chain alcohol dehydrogenase which is also called type II hydroacylcoA dehydrogenase, 17β-hydrosteroid dehydrogenase type 10 and 2-methyl-3 hydroxybutyrylcoA dehydrogenase. ABAD acts on various substrates such as branched chain fatty acids, alcohols, amino acid catabolites and steroids. In the brain, it is primarily localized in mitochondria of neurons. Previous studies have shown high ABAD expression in the temporal lobe and hippocampus of AD affected patients (88;89).

Animals with transgenic (Tg) APP mice have also demonstrated higher expression of ABAD (88). As the name suggests, ABAD directly binds to Aβ protein which is highly expressed in Alzheimer's patients. These links suggest role of ABAD dehydrogenase in the pathophysiology of Alzheimer's disease. Another crucial factor in causing AD as discussed earlier is mitochondrial dysfunction, related oxidative stress and hypometabolism. In recent studies, it was hypothesized that ABAD can act as a crucial link between increased Aβ production and mitochondrial dysfunction in Alzheimer's disease progression (90-92).To test this hypothesis, double transgenic mice with increased levels of ABAD and Aβ were developed (along with Tg mAP, Tg ABAD, and not Tg littermate controls)(93). Neuron cultures derived from these Tg mice showed increased ROS, oxidative stress and relative decrease in ATP production. Further studies indicated defective activity of mitochondrial Complex IV as the source of the ROS species, also such effect was not observed in single Tg mice with increased ABAD alone (suggesting Aβ acting as a crucial element linking the two). Lactate dehydrogenase (LDH) was higher in the Tg mAP/ABAD mice as compared to other groups suggesting the reversal to lactate metabolism. Cell apoptosis via caspase 3 activity was observed in *in vitro* studies. Data from the Tg mice also suggested reduced ATP production at 9 months of age along with reduced Complex IV activity. Overall, ABAD acts a crucial enzyme that can lead to mitochondrial dysfunction and disease progression in AD.

104 Dehydrogenases

important role in pathogenesis of AD.

mutations in APP protein or ApoE protein (a protein linked to lipid and cholesterol in the body) as well as Down's syndrome are some examples of genetic predispositions that increase the propensity to develop Alzheimer's disease. However, the etiology of the disease is still questionable. Both vascular as well as metabolic dysfunction have been accredited as major factors prodromic to the pathogenesis and progression of Alzheimer's disease (84). Vascular dysfunction includes reduction in cerebral blood flow, reduced glucose uptake, and reduced amyloid beta clearance with cerebral amyloid angiopathy. The patients with atherosclerosis have shown to have an increased risk to develop AD. Vascular structural anomalies in cerebral vessels (like increased pinocytic activity as well as swelling) have been seen in early AD further signifying the importance of vascular dysfunction in AD pathogenesis. Defective glucose utilization has been observed earlier than reduced cerebral blood flow (CBF), indicating the role of glucose metabolism in AD development. What is of primary interest to us is that it is slowly becoming well accepted that metabolic dysfunction, related oxidative stress and mitochondrial deficits can precede AD development (84-86). In one such recent study, triple transgenic mice 3xTg-AD were developed (having mutations in human APPSWE, TauP301L, PS1M146V genes linked with AD) along with their respective controls. Decreased mitochondrial respiration was observed along with reduced PDH activity. High levels of oxidative stress (via measurement of hydrogen peroxide production and related lipid peroxidation), high Aβ levels with high levels of Aβ binding alcohol dehydrogenase (ABAD) was observed. Decreased respiration was also observed in embryonic neurons, which continued till senescence leading to AD pathogenesis. Thus these studies clearly emphasized how mitochondrial dysfunction and resulting metabolic respiration preceded AD development. Defective enzyme function(of dehydrogenases) in pathways of energy metabolism such as glycolysis, tricarboxylic acid pathway and electron transport chain have been well studied in AD progression. Defective PDH, KGDHC, cytochrome oxidase along with reduced activity of hexokinase, phosphofructokinase are the major enzymes reported in AD so far (87). In the paragraph below we will be further discuss some of these DHOs and their mechanism of metabolic dysfunction in AD. We will also look at other DHOs apart from those directly involved in the bioenergetics which have an

One such DHO as mentioned earlier is Aβ binding alcohol dehydrogenase (ABAD). ABAD is a short-chain alcohol dehydrogenase which is also called type II hydroacylcoA dehydrogenase, 17β-hydrosteroid dehydrogenase type 10 and 2-methyl-3 hydroxybutyrylcoA dehydrogenase. ABAD acts on various substrates such as branched chain fatty acids, alcohols, amino acid catabolites and steroids. In the brain, it is primarily localized in mitochondria of neurons. Previous studies have shown high ABAD expression

Animals with transgenic (Tg) APP mice have also demonstrated higher expression of ABAD (88). As the name suggests, ABAD directly binds to Aβ protein which is highly expressed in Alzheimer's patients. These links suggest role of ABAD dehydrogenase in the pathophysiology of Alzheimer's disease. Another crucial factor in causing AD as discussed earlier is mitochondrial dysfunction, related oxidative stress and hypometabolism. In recent

in the temporal lobe and hippocampus of AD affected patients (88;89).

**Figure 6.** : Correlation between aging free radicals, DHOs and the onset of CNS disorders.

In another study, ABAD-decoy peptide (ABAD-DP) was introduced in the Tg mAPP mice, which prevented the interaction of ABAD with Aβ. As expected, reduction in ABAD-Aβ complex formation accompanied with attenuated oxidative stress, increased oxygen consumption, increased activity of enzymes associated with mitochondrial respiratory chain, improvement in energy metabolism, and increased spatial memory (89). Thus based on these studies, inhibitors of ABAD-Aβ hold promise as potential targets for the treatment of AD.

Role and Function of Dehydrogenases in CNS and Blood-Brain Barrier Pathophysiology 107

cause of PD. It is also characterized by accumulation of α-synuclein into inclusions called Lewy bodies. 60% of DA neurons are dead and 70% responsiveness of DA is lost. Mostly PD is idiopathic, however specific genetic mutations have shown to increase the risk to develop PD. Mutations is genes such as α-synuclein, Parkin, PINK1 have been reported so far. After diagnosis of PD based on its classical symptoms and neuroimaging, treatment is usually done using levodopa (L-DOPA). L-DOPA is converted to dopamine in the brain and can temporarily alleviate the motor symptoms. Dopamine receptor agonists as well as selective monoamine oxidase-B (MAO-B) inhibitors are also administered along with L-DOPA (97;98). Treatment thus helps to partially reduce the symptoms of PD, since the actual underlying cause of this disease is still unknown. Altered enzyme activity and

Aldehyde dehydrogenase plays an important role in detoxifying aldehydes in brain. Reduced expression of isoforms of ALDH such as ALDH1A1 and ALDH2 is reported in PD patients. In addition impaired Complex I activity is documented in PD which can reduce the availability of NAD+ cofactor required by ALDHs to remove toxic biogenic aldehydes. Thus decreased ALDH function could be the underlying factor preceding the development of PD. Using transgenic mice null for both ALDH1A1 and ALDH2, the risk to develop PD was tested (99). Such mice exhibited deficits in motor performance typical of PD. Loss of DA with increased accumulation of biogenic aldehydes such as HNE was observed. L-DOPA administration alleviated the motor deficits suggesting a role of ALDHs in the

Another DHO implicated in PD is glutamate dehydrogenase (GDH). GDH is a key enzyme involved in interconversion of glutamate to alpha-ketoglutarate and ammonia using NADP(H) and NAD(H) as co factors. It plays an important role in homeostasis by interconnecting amino acid and carbohydrate metabolism pathways. Present in two isoforms in humans, the GDH isoform 2 (hGDH2) is overexpressed in the brain astrocytes and the sertoli cells in testis. ADP levels act as positive regulators for this enzyme and unlike the other isoform it is not inhibited by GTP. Important in recycling glutamate in the brain astrocytes, this enzyme works in concert with glutamine synthetase (GS) providing ammonia as well as ATP for GS activity. Two parallel studies have shown that increased levels of glutamate prepones the onset of the disease by 6 to 13 years (100). Hemizygous individuals with a rare variation in hGDH2 (substitution of Ala for Ser445) was detected in these individuals. GDH deficiencies have also been linked to the onset of epilepsy. All together these results highlight the role of hGDH2 in the maintenance of brain homeostasis.

ABAD associated with Alzheimer's disease has also shown to play some role in PD disease. ABAD expression is seen to be downregulated in PD patients (101). In mouse models of PD generated by administration of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) ABAD expression is significantly reduced. By contrast, overexpression of ABAD in transgenic mice is shown to attenuate MPTP-induced dopaminergic neurogeneration. This strongly suggests that ABAD may contribute to the fate of DA neurons during the onset of

mitochondrial dysfunction has been linked to PD as well.

pathophysiology of PD.

PD.

Another dehydrogenase that is implicated in AD progression is aldehyde dehydrogenase (ALDH) (89;94). Aldehyde dehydrogenase is observed as a key enzyme in the brain involved in metabolism and degradation of biogenic aldehydes, monoamine neurotransmitters such as norepinephrine, dopamine, diamines and GABA. Recent studies have also shown that patients with Down's syndrome have reduced activity of ALDH enzyme (95). Two dimensional analysis of proteins extracted from brain samples of nine aged patients with Down's syndrome and nine controls showed that ALDH was down regulated in the patients with Down's syndrome. This resulted in accumulation of aldehydes and further formation of tangles and plaques as observed in aged patients with Down's syndrome.

Oxidative stress and generation of ROS species has been implicated in Alzheimer's disease as elaborated earlier. These oxygen species modify proteins, nucleic acids as well as lead to lipid peroxidation. Lipid peroxidation produces toxic aldehydes such as 4-hydroxy-2-nonenal (HNE) in several disorders such as Alzheimer's as well as Parkinson's disease. In the brain, normally ALDH2- an isoform of aldehyde dehydrogenase oxidizes and degrades end product of lipid peroxidation such as HNE. The role of ALDH in oxidative stress and age dependent memory loss and decline in cognitive function was studied using a transgenic mouse model with defective ALDH2 (96). A dominant negative form of ALDH2 mice was produced and its effect on the metabolic pathways as well as accumulation of toxic products was tested. As expected HNE accumulation was observed in such transgenic mice compared to controls. Further testing of cognitive capability was performed using object recognition and water maze test. Decreased cognitive function in the transgenic mice was observed along with accumulation of tau phosphorylation (a typical pathological sign of Alzheimer's disease).

A dominant negative form of ALDH2 mice was produced and its effect on the metabolic pathways as well as accumulation of toxic products was tested. As expected HNE accumulation was observed in such transgenic mice compared to controls. Further testing of cognitive capability was performed using object recognition and water maze test. Decreased cognitive function in the transgenic mice was observed along with accumulation of tau phosphorylation (a typical pathological sign of Alzheimer's disease).
