Genetics of AD and PD 705 mouse models with missense mutations of the endogenous murine PSEN1 and high Aβ42 levels perform poorly on the object recognition test (Huang et al., 2003; Janus et al., 2000). Double PSEN1/APP transgenics have been developed and suggest that PSEN1, APP, and mutations within these genes, play a role in the production of Aβ (Holcomb et al., 1998; Mineur et al., 2005). Genetic Variation To date, there have been 123 PSEN1 mutations reported (Fig. 3). A com- prehensive list of PSEN1 mutations is available through the NCBI database (http://www.molgen.ua.ac.be/ADmutations). The majority of these mutations are missense mutations. These missense mutations cause amino acid substitutions throughout the PSEN1 protein and appear to result in a relative increase in the ratio of the Aβ42 to Aβ40 peptides via either increased Aβ42 or decreased Aβ40 generation, or a combination of both (Scheuner et al., 1996). For example, individ- uals that carry the PSEN1 L166P mutation can have an age-at-onset in adolescence, Fig. 3 AD3: PSEN1 structure and mutations. Thus far, at least 123 mutations in the PSEN1 gene have been described, of which a few are shown. For a more complete list of PSEN1 mutations, see http://www.molgen.ua.ac.be/ADMutations. TM, transmembrane domains. Scale is approximate 706 L.M. Bekris et al. and in vitro studies indicate that this mutation induces exceptionally high levels of Aβ42 production as well as impairs notch intracellular domain production and notch signaling (Moehlmann et al., 2002). AD4: Presenilin 2 Inheritance and Clinical Features A candidate gene for the chromosome 1 AD4 locus was identified in 1995 in a Volga German AD kindred with a high homology to the AD3 locus (PSEN1) and was later named presenilin 2 (PSEN2) (Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1996). In contrast to mutations in the PSEN1 gene, missense mutations in the PSEN2 gene are a rare cause of EOFAD, at least in Caucasian pop- ulations. The age of onset in PSEN2-affected families appears to be older (45–88 years) than that observed in PSEN1-affected families (25–65 years). Age of onset is highly variable among PSEN2-affected family members within the same family, whereas for PSEN1-affected families, the age of onset is generally quite similar among affected family members and is even similar among members of differ- ent families with the same mutation (Campion et al., 1999; Rogaev et al., 1995; Sherrington et al., 1996, 1995). Missense mutations in the PSEN2 gene may be of lower penetrance than PSEN1 mutations and thus be subject to the modifying action of other genes or environmental influences (Sherrington et al., 1996; Tandon and Fraser, 2002). Gene Location and Structure The PSEN2 gene is located on chromosome 1 (1q42.13) and was identified by sequence homology and cloned (Levy-Lahad et al., 1995; Rogaev et al., 1995). PSEN2 has 12 exons and is organized into 10 translated exons that encode a 448 amino acid peptide. The PSEN2 protein is predicted to consist of 9 transmembrane domains and a large loop structure between the sixth and seventh domains (Fig. 4). PSEN2 also displays tissue-specific alternative splicing (ADCG, 1995; Anwar et al., 1996; Hutton et al., 1996; Levy-Lahad et al., 1995; Prihar et al., 1996; Rogaev et al., 1995). Gene Function and Expression Like PSEN1, PSEN2 has been described as a component of the atypical aspartyl protease called γ-secretase that is responsible for the cleavage of Aβ (De Strooper et al., 1998; Wolfe et al., 1999b). PSEN2-associated mutations have been reported to increase the ratio of Aβ42 to Aβ40 (Aβ42/Aβ40) in mice and humans (Citron et al., 1997; Scheuner et al., 1996), indicating that presenilins might modify the way in which γ-secretase cuts APP. APP processing at the gamma-secretase site has been reported to be affected in variable ways by the presenilin mutations. For example, PSEN1-L166P mutations cause a reduction i n Aβ production whereas the PSEN1-G384A mutant significantly increases Aβ42. In contrast, PSEN2 appears to be a less efficient producer of Aβ than PSEN1 (Bentahir et al., 2006). The functions and biological importance of presenilin splice variants are poorly understood. But Genetics of AD and PD 707 Fig. 4 AD4: PSEN2 structure and mutations. Thus far, at least 16 mutations in the PSEN2 gene have been described, of which a few are shown. For a more complete list of PSEN2 mutations, see http://www.molgen.ua.ac.be/ADMutations. The V393M novel mutation was most recently found in one case (Lindquist et al., 2008). TM, transmembrane domains. Scale is approximate it appears that differential expression of presenilin isoforms may lead to differential regulation of the proteolytic processing of the APP protein. For example, aberrant PSEN2 transcripts lacking exon 5 increase the rate of production of Aβ peptide (Sato et al., 2001), whereas naturally occurring isoforms without exons 3 and 4 and/or without exon 8 do not affect production of Aβ (ADCG, 1995; Grunberg et al., 1998). PSEN2 is expressed in a variety of tissues, including the brain where it is expressed primarily in neurons (Kovacs et al., 1996). Genetic Variation Mutations in PSEN2 are a much rarer cause of FAD than are PSEN1 mutations, having been described in only six families, including the Volga German kindred where a founder effect has been demonstrated (Cruts and Van Broeckhoven, 1998; Levy-Lahad et al., 1995; Rogaev et al., 1995; Sherrington et al., 1996). To date, as many as 16 PSEN2 mutations have been identified. One of the first mutations to be identified was a point mutation resulting in the substitution of an isoleucine for an asparagine at residues 141 (N141l) located within the second transmembrane domain (Levy-Lahad et al., 1995). Most recently, a V393M mutation located within the seventh transmembrane domain has been described (Lindquist et al., 2008) (Fig. 4). A comprehensive list of PSEN2 mutations is available through the NCBI database (http://www.molgen.ua.ac.be/ADmutations). 708 L.M. Bekris et al. 1.2.3 Genes Associated with Risk in Sporadic Alzheimer’s Disease AD2: APOE Inheritance and Clinical Features The APOE gene has been associated with both familial late-onset and sporadic late- onset AD in numerous studies of multiple ethnic groups. There are three major protein isoforms of human apoE (apoE2, apoE3, and apoE4), which are the prod- ucts of three alleles (2, 3, and 4). The frequency of the APOE 4 allele varies between ethnic groups, but the APOE 4– carriers are the most frequent in controls across all ethnic groups and APOE 4+ carriers are the most frequent in AD patients (Brousseau et al., 1994; Chauhan, 2003; Farrer et al., 1997, 1995; Hendrie et al., 1995; Liddell et al., 1994; Lucotte et al., 1994; Mayeux et al., 1993; Poirier et al., 1993; Roses et al., 1995; Schellenberg, 1995; Selkoe, 2001;Tsaietal.,1994). The APOE 4 genotype is associated with higher risk of AD (Corder et al., 1993), with earlier age of onset of both AD (Tang et al., 1996) and Down syndrome (where there is an additional copy of chromosome 21 carrying the APP gene) (Schupf and Sergievsky, 2002), and also with a worse outcome after head trauma (Nicoll et al., 1995) and stroke, both in humans (Liu et al., 2002b) and in transgenic mice expressing human apoE4 (Horsburgh et al., 2000). Gene Location and Structure The APOE gene is located on chromosome 19q13.2 and consists of 4 exons that encode a 299 amino acid protein. The APOE gene is in a cluster with other apolipoprotein genes: APOC1, APOC2, and APOC4.TheAPOE 4 loci are located within exon 4 of the gene. The three APOE 4 alleles (2, 3, and 4) defined by two single nucleotide polymorphisms, rs429358 and rs7412, encode three protein isoforms (E2, E3, and E4). The most frequent isoform is apoE3, which contains cysteine and arginine at amino acid positions 112 and 158. Both positions contain cysteine residues in apoE2 and arginine residues in apoE4 (Fig. 5). This substitu- tion affects the three-dimensional structure and the lipid-binding properties between isoforms. In apoE4, the amino acid substitution results in a changed structure with the formation of a salt-bridge between an arginine in position 61 and a glutamic acid in 255 that causes this isoform to bind preferentially to VLDL whereas apoE3 and apoE2 bind preferentially to high-density lipoproteins (HDLs) (Mahley et al., 2006). Gene Function and Expression The mechanisms that govern apoE toxicity in the brain are not fully understood. Some proposed mechanisms include isoform specific toxicity, apoE E4–mediated amyloid aggregation, and apoE E4–mediated tau hyperphosphorylation (Huang, 2006). It is known that apoE plays an important role in the distribution and metabolism of cholesterol and triglycerides within many organs and cell types in the human body (Mahley et al., 2006). The apoE polymorphism is unique to humans and has been Genetics of AD and PD 709 Fig. 5 AD2: APOE structure and single nucleotide polymorphisms (SNPs). The general protein structure of apoE is shown (panel a). The two SNPs and corresponding protein locations are shown (rs429358 and rs7412; C112R and R158C). The APOE 2, 3, 4 haplotype is shown in panel b. Receptor binding domain; R. Scale is approximate proposed to have evolved as a result of adaptive changes to diet (Finch and Stanford, 2004; Mahley and Rall, 1999). Individuals carrying APOE 4 have higher total and LDL cholesterol (Sing and Davignon, 1985). Neurons, in vitro, have a cholesterol uptake that is lower when the lipid is bound to apoE4 compared to apoE2 and apoE3 (Rapp et al., 2006), and apoE4 appears to be less efficient than the other isoforms in promoting cholesterol efflux from both neurons and astrocytes (Michikawa et al., 2000). Chylomicron remnants and very low density lipoprotein (VLDL) remnants are rapidly removed from the circulation by receptor-mediated endocytosis. ApoE, the major apolipoprotein of the chylomicron in the brain, binds to a specific receptor and is essential for the normal catabolism of triglyceride-rich lipoprotein con- stituents. Defects in apolipoprotein E result in familial dysbetalipoproteinemia, or type III hyperlipoproteinemia (HLP III), in which increased plasma cholesterol and triglycerides are the consequence of impaired clearance of chylomicron and VLDL remnants (Mahley et al., 1999). In the brain, lipidated apoE binds aggregated Aβ in a isoform-specific manner, apoE4 being much more effective than the other forms, 710 L.M. Bekris et al. and has been proposed to enhance deposition of the Aβ peptide (Stratman et al., 2005). Brain cells from APOE knock-out mice (APOE−/−) are more sensitive to exci- totoxic and age-related synaptic loss (Buttini et al., 1999), whereas Aβ-induced synaptosomal dysfunction is also enhanced compared to control animals (Keller et al., 2000). When human apoE isoforms are expressed i n APOE−/− mice, the expression of apoE3, but not apoE4, is protective against age-related neurodegen- eration (Buttini et al., 1999) and Aβ toxicity (Keller et al., 2000). In addition, astrocytes, from APOE−/− mice that express human apoE3, release more choles- terol than those expressing apoE4, suggesting that apoE isoforms may modulate t he amount of lipid available for neurons. Other studies report apoE-specific effects on Aβ removal from the extracellular space whereby the apoE3 isoform has a higher Aβ binding capacity than ApoE4 when associated with lipids (Canevari and Clark, 2007; LaDu et al., 1995). In humans the greatest expression of apoE is found in the liver, followed by the brain. Animal and in vitro models show that in the brain, astrocytes and microglia are the main producers of secreted apoE (Pitas et al., 1987; Uchihara et al., 1995) whereas neurons appear to produce apoE under stress conditions (Aoki et al., 2003; Xu et al., 1999). In a rodent model, moderate injury induces enhancement of apoE levels in clusters of CA1 and CA3 pyramidal neurons (Boschert et al., 1999); in another model, apoE levels increase in response to peripheral nerve injury (Ignatius et al., 1986) whereas apoE secretion in human primary astrocytes can be reduced by a combination cytokines (Baskin et al., 1997). In addition, individuals carrying apoE4 have higher amyloid and tangle pathol- ogy (Nagy et al., 1995), and they have an increase in mitochondrial damage (Gibson et al., 2000) compared to those carrying other forms. Genetic Variation The gene dose of APOE 4 is a major risk factor for the disease, with many stud- ies reporting an association between gene dose, age-at-onset (Blacker et al., 1997), and cognitive decline (Martins et al., 2005). After age 65, the risk among family members increases depending on the number of 4 alleles present in the affected individual. Risks to family members with the APOE 2/2 and 2/3 genotypes are nearly identical at all ages to risks for family members with the APOE 3/3 genotype. Among family members with APOE 3/3, the lifetime risk for AD by age 90 can be as much as three times greater than the expected proportion of APOE 4 carriers, suggesting that factors other than APOE contribute to AD risk. In addition, a 44% risk of AD by age 93 among family members of APOE 4/4 carriers indicates that as many as 50% of people having at least one e4 allele do not develop AD. There also appears to be a gender modification effect because the risk to male family members with APOE 3/4 is similar to that for the APOE 3/3 group but significantly less than the risk for the APOE 4/4 carriers; whereas among female family members the risk for the APOE 3/4 carriers is nearly twice that for the APOE 3/3 carriers (Brousseau et al., 1994; Farrer et al., 1997, 1995; Hendrie et al., 1995; Liddell et al., 1994; Lucotte et al., 1994; Mayeux et al., 1993; Poirier et al., 1993;Tsaietal.,1994). Genetics of AD and PD 711 1.3 Summary AD is characterized by an irreversible progressive loss of memory and cognitive skills that can occur in rare familial cases as early as the third decade. Currently there is no cure for AD, and treatments only slow AD progression slightly in some patients (Raina et al., 2008; Raschetti et al., 2007). The early-onset familial forms of AD have an autosomal dominant inheritance linked to three genes: APP, PSEN1, and PSEN2. The most common sporadic form of AD occurs after the age of 60 and has thus far been consistently, across numerous studies, associated with only one gene, the APOE gene. The mechanistic contribution of these genes in AD pathogenesis has been studied extensively but the specific biology involved in the progression of AD remains unclear, suggesting that AD is a genetic and environmentally complex disease. 2 Parkinson’s Disease 2.1 Introduction 2.1.1 Prevalence and Incidence Parkinson’s disease (PD) (OMIM #168600) is the second most common neu- rodegenerative disorder. The incidence is similar worldwide, with the prevalence increasing in proportion to regional increases in population longevity with more than 1% affected over the age of 65 years and more than 4% of the popula- tion affected by the age of 85 years (de Rijk et al., 2000). Idiopathic PD is the most frequent form of Parkinsonism and accounts for over 75% of all PD cases, and it usually refers to a s yndrome characterized by late-onset, largely non- genetic movement disorder (Gibb and Lees, 1988). Rare forms of PD in which genetic factors dominate, represent 5–10% of all PD patients (Belin and Westerlund, 2008). 2.1.2 Clinical Symptoms Clinical manifestations that can be detected by neurological examinations include tremor, rigidity, bradykinesia, and postural instability. Disruption of motor abilities is associated with striatal dopamine levels thought to arise from selective and pro- gressive loss of dopaminergic cells within the substantia nigra pars compacta and the locus ceruleus of the midbrain (Tan and Skipper, 2007). Secondary symptoms may involve cognitive dysfunction and subtle language problems. Symptoms can be both chronic and progressive. Levodopa remains the most effective treatment of PD symptoms but its use is complicated by the emergence of motor fluctuations and dyskinesias. Dopamine agonists, catechol-O-methyltransferase inhibitors, and other anti-Parkinsonian drugs may diminish or prevent these complications and possibly exert disease-modifying effects (Jankovic, 2006). 712 L.M. Bekris et al. 2.1.3 Clinical Diagnosis Diagnostic clinical criteria of PD include four cardinal symptoms: bradykinesia, rest tremor, rigidity, and postural instability. An additional criterion includes a therapeutic response of tremor to levodopa (Galpern and Singhal, 2006). In addi- tion, other common motor signs and symptoms include loss of automatic motor movements such as loss of arm swing, loss of blinking, and difficulty in perform- ing simultaneous motor acts. Many nonmotor symptoms can also be present in PD, such as cognitive impairment, hallucination, delusion, behavioral abnormali- ties, clinical depression, disturbances of sleep and wakefulness, loss of smell, pain, and autonomic dysfunctions such as constipation, hypotension, urinary frequency, impotence, and sweating (Mizuno et al., 2008). 2.1.4 Neuropathological Diagnosis The diagnosis of idiopathic PD may also involve confirmation upon autopsy where neuropatholgical assessment of the amount of neuronal loss and Lewy-related pathology (Lewy bodies and Lewy neurites), in the brainstem and elsewhere in the brain, is performed. Eosinophilic neuronal cytoplasmic inclusions known as Lewy bodies (LBs) are found in PD postmortem brain (Gibb and Lees, 1988). The disease is also characterized by dopamine neuron degeneration and depigmentation of the substantia nigra accompanied by neuronal loss in other brainstem regions including the ventral tegmental area and locus ceruleus (Belin and Westerlund, 2008; Love, 2005). The principal component of LBs is α-synuclein, and LBs are best visualized immunohistochemically, using an antibody to α-synuclein (Love, 2005). The func- tion of α-synuclein is unknown. I t is primarily found in neural tissue in presynaptic terminals. It can also be found in glial cells. It is predominantly expressed in the neo- cortex, hippocampus, substantia nigra, thalamus, and cerebellum (George, 2002). LBs are typically found in the substantia nigra and locus ceruleus, where there is substantial neuronal loss and gliosis. LBs may also be found in the dorsal motor nucleus of the vagus where LBs are usually roughly spherical, with an eosinophilic core surrounded by a paler ‘‘halo.’’ Within the cerebrum, LBs are usually present in the amygdaloid nuclei, parahippocampal and cingulate gyri, and insula, but they may also be found in other parts of the neocortex. The cholinergic nucleus basalis of Meynert may also be affected. Cortical LBs appear as regions of homoge- neous eosinophilic staining of neuronal cytoplasm and eccentric displacement of the nucleus (Love, 2005). Lewy neurites are nerve cell processes that contain aggregates of α-synuclein and are most numerous in the CA2/3 region of the hippocampus and in the substantia nigra (Love, 2005). 2.2 Genetics of Parkinson’s Disease 2.2.1 Introduction Historically, PD was considered to be largely sporadic in nature without genetic origin. However, in the past decade, genetic studies of PD families from different Genetics of AD and PD 713 geographical regions worldwide have strengthened the hypothesis that PD has a substantial genetic component. One of the first autosomal dominant inherited forms of PD was identified in an Italian family, and it is named PARK1 ( Polymeropoulos et al., 1996). Since then, 13 loci, PARK1–13, have been linked to rare forms of PD: autosomal dominant and autosomal recessive PD (Belin and Westerlund, 2008; Farrer, 2006). Of these 13 loci, eight genes have been described as causing PD: four autosomal dominant (SNCA, LRRK2, UCHL1, and HTRA2) and four autosomal recessive (PRKN, DJ1, PINK1, and ATP13A2; Table 1). Mutations in the SNCA, LRRK2, PRKN, and PINK1 genes are the most well-chararacterized as causing PD whereas mutations in the other genes listed do not have as much supporting evidence as causes of PD. Recently, a clinical association has been reported between PD and type-1 Gaucher’s disease, which is caused by a glucocerebrosidase deficiency owing to mutations in the glucocerebrosidase gene (GBA), and several studies have found an association between GBA mutations and PD (Aharon-Peretz et al., 2004;Bras et al., 2007; Clark et al., 2005, 2007; De Marco et al., 2008; Eblan et al., 2006; Gan-Or et al., 2008; Lwin et al., 2004; Sato et al., 2005; Spitz et al., 2008;Tan et al., 2007;Toftetal.,2006; Wu et al., 2007; Ziegler et al., 2007). The GBA gene has not yet been named as a PD gene but is described briefly here. Some PD genes where mutations have been linked to familial forms of PD are also candidate genes for sporadic forms of PD, as those genes (SNCA and LRRK2) may also carry other mutations that merely increase risk (Table 1). 2.2.2 Genes Associated with Autosomal Dominant Parkinson’s Disease PARK1 and PARK4: SNCA Inheritance and Clinical Features PARK1- and PARK4-linked PD are both of autosomal dominant inheritance, but PARK1 is caused by missense mutations in the α-synuclein gene (SNCA) and PARK4, by multiplications of SNCA. Affected family members are mostly of juvenile-onset with atypical clinical features including myoclonus and hypoven- tilation, with rapid progression of symptoms. Three missense mutations, A53T (Polymeropoulos et al., 1996), A30P (Kruger et al., 1998), and E46K (Zarranz et al., 2004); duplications (Chartier-Harlin et al., 2004 ; Fuchs et al., 2007; Ibanez et al., 2004; Nishioka et al., 2006); and triplications (Farrer et al., 2004; Singleton et al., 2003)ofSNCA are known (Fig. 6). The A53T substitution was Fig. 6 PARK1 and PARK4: SNCA structure and mutations. The general protein structure of α- synuclein is shown (Bisaglia et al., 2008). Scale is approximate 714 L.M. Bekris et al. the first mutation identified in a large family with autosomal dominant disease (Polymeropoulos et al., 1996). Later, A30P and E46K substitutions were identi- fied in a German and a Spanish family, respectively, with clinical features described as dementia with LB (Kruger et al., 1998; Zarranz et al., 2004). PARK1 missense mutations and PARK4 multiplications are both extremely rare causes of familial Parkinsonism (Chartier-Harlin et al., 2004; Farrer et al., 2004; Fuchs et al., 2007; Ibanez et al., 2004; Nishioka et al., 2006; Singleton et al., 2003). Gene Location and Structure SNCA is located on chromosome 4q22.1, has six exons, and encodes a 140 amino acid protein. The N-terminus consists of an amphipathic α-helical domain that asso- ciates with membrane microdomains, known as lipid rafts (Fortin et al., 2004). The central region contains a fibrillization region, and the C-terminus contains an aggregation inhibition region (Fig. 6) (Bisaglia et al., 2008). Gene Function and Expression SNCA is expressed throughout the mammalian brain and is enriched in presynaptic nerve terminals (George, 2002). The protein can adopt partially folded structures but in its native form is unfolded and can assume both monomeric and oligomeric alpha helix and beta-sheet conformations, as well as morphologically diverse aggre- gates, ranging from those that are amorphous to amyloid-like fibrils (Uversky, 2003). These fibrillar moieties are a component of LBs in both familial and idio- pathic PD (Spillantini et al., 1997), but it is unclear whether the fibrils themselves, or the oligomeric fibrilization intermediates (protofibrils), are toxic to the cell. Interestingly, SNCA genomic multiplications in familial PD are associated with an increase in protein expression (Farrer et al., 2004) and brain samples of triplication mutant carriers show protofibril formation is enhanced with an increase in SNCA expression (Miller et al., 2004). In vitro, A30P, A53T, and E46K mutant proteins show an increased propensity for self-aggregation and oligomerization into protofib- rils, compared with wild-type protein (Conway et al., 1998; Pandey et al., 2006) that may be related to the membrane permeabilization activity of these protofib- rils, which form pore-like and tubular structures (Lashuel et al., 2002). It appears that only A53T and E46K promote formation of the fibrils (Conway et al., 2000; Greenbaum et al., 2005) whereas A30P has been reported to disrupt the interaction between α-synuclein and the lipid raft and to possibly redistribute the protein away from the synapse (Fortin et al., 2004). A mouse spontaneous deletion strain is viable, fertile, and phenotypically nor- mal (Specht and Schoepfer, 2001) whereas overexpression of wild-type SNCA in a mouse model has many features of PD, such as loss of dopaminergic terminals in the striatum, mislocalization and accumulation of insoluble α-synuclein, and motor abnormalities (Rockenstein et al., 2002; Fleming et al., 2004; Masliah et al., 2000 ). Both A30P and A53T mutant mouse models display neuronal cell loss and motor changes (Melrose et al., 2006). . protein isoforms (E2, E3, and E4). The most frequent isoform is apoE3, which contains cysteine and arginine at amino acid positions 112 and 158. Both positions contain cysteine residues in apoE2. peripheral nerve injury (Ignatius et al., 1986) whereas apoE secretion in human primary astrocytes can be reduced by a combination cytokines (Baskin et al., 1997). In addition, individuals carrying apoE4. Lewy neurites), in the brainstem and elsewhere in the brain, is performed. Eosinophilic neuronal cytoplasmic inclusions known as Lewy bodies (LBs) are found in PD postmortem brain (Gibb and Lees,