Anna Anvret

Developmental regulation of leucine-rich repeat kinase 1 and 2 expression in the brain and other rodent and human organs: Implications for Parkinson’s disease

Mutations in leucine-rich repeat kinase 2 (LRRK2) constitute the most common known cause of Parkinsons disease (PD), accounting for both familial and sporadic forms of the disease. We analyzed the tempo-spatial activity of leucine-rich repeat kinase 1 (LRRK1) and LRRK2 at the cellular level in human and rat tissues including development and aging. Lrrk2 mRNA is expressed in adult rat striatum, hippocampus, cerebral cortex, sensory and sympathetic ganglia, lung, spleen and kidney. In the developing rat striatum, Lrrk2 transcription is first observed at postnatal day (P) 8 followed by increasing mRNA levels during the following 3 weeks, as revealed by quantitative in situ hybridization, after which levels remain up to 24 months of age. The time-course of postnatal development of Lrrk2 activity in striatum thus closely mirrors the postnatal development of the dopamine innervation of striatum. Lrrk2 mRNA is seen in P1 rat lung, heart, and kidney, whereas Lrrk1 is found in many areas of the P1 rat. Lrrk1 is present in adult rat brain, adrenal gland, liver, lung, spleen and kidney and also in embryonic brain, with declining gene activity after birth. LRRK1 and LRRK2 are active in the adult human cortex cerebri, hippocampus and LRRK2, but not LRRK1, in striatum. Transcription of both genes is also seen in the young human thymus and LRRK2 is active in tubular parts of the adult human kidney. Our findings suggest that the two paralogous genes have partly complementary expression patterns in the brain, as well as in certain peripheral organs including lymphatic tissues. While the strong presence of Lrrk2 message in striatum is intriguing in relation to PD, the many other neuronal and non-neuronal sites of Lrrk2 activity also needs to be taken into account in deciphering possible pathogenic pathways.

Expression of PINK1 mRNA in human and rodent brain and in Parkinson's disease

Mutations in PINK1 (PTEN-induced putative kinase 1) are causal for early onset recessive parkinsonism in humans, characterized by damage to the nigrostriatal system. In situ hybridization studies in rodent brains have suggested a predominantly neuronal expression of PINK1 mRNA but immunocytochemistry of human brain tissue has shown PINK1-like immunoreactivity in both neurons and glia. In this study, we assessed the comparative distribution of PINK1 mRNA in human, rat and mouse brain. We observe that in humans PINK1 message is expressed in neurons with very little to no signal in glia and confirms similar findings in rodent tissue. Highest levels of expression were observed in hippocampus, substantia nigra and cerebellar Purkinje cells. We also show that PINK1 mRNA expression is similar in nigral neurons from neurologically normal controls and sporadic Parkinsons disease cases.

Association of a polymorphism in the ABCB1 gene with Parkinson's disease.

The ATP-binding cassette, sub-family B, member 1 (ABCB1) gene encoding the protein P-glycoprotein (P-gp) has been implicated in the pathophysiology of Parkinsons disease (PD) due to its role in regulating transport of endogenous molecules and exogenous toxins. In the present study, we analyzed the ABCB1 single nucleotide polymorphisms (SNPs) 1236C/T (exon 12), 2677G/T/A (exon 21) and 3435C/T (exon 26) in 288 Swedish PD patients and 313 control subjects and found a significant association of SNP 1236C/T with disease (p=0.0159; chi(2)=8.28), whereas the distributions of wild-type and mutated alleles were similar for 2677G/T/A and 3435C/T in patients and controls. Haplotype analysis revealed significant association of the 1236C-2677G haplotype with PD (p=0.026; chi(2)=4.955) and a trend towards association with disease of the 1236C-2677G-3435C haplotype (p=0.072; chi(2)=3.229). Altered ABCB1 and/or P-pg expression was recently shown in PD patients, and impaired drug efflux across barriers such as the gastrointestinal and nasal mucosal linings or the blood-brain barrier, might result in accumulation of drugs and/or endogenous molecules in toxic amounts, possibly contributing to disease. ABCB1 polymorphisms thus constitute an example of how genetic predisposition and environmental influences may combine to increase risk of PD.

Cerebellar α-synuclein levels are decreased in Parkinson’s disease and do not correlate with SNCA polymorphisms associated with disease in a Swedish material

Alterations of brain and plasma α‐synuclein levels and SNCA gene variability have been implicated in the pathogenesis of Parkinsons disease (PD). We therefore measured α‐synuclein protein levels in postmortem PD and control cerebellum tissue using Western blot and investigated whether the levels correlated to SNCA genotype. We found markedly decreased α‐synuclein levels in PD patients (n=16) compared to gender‐and age‐matched controls (n=14; P= 0.004) normalized to α‐tubulin. We also performed an association study of the noncoding polymorphisms rs2737029 (A/G) and rs356204 (A/G) (intron 4), and of rs356219 (T/C) (34′‐region) of SNCAin a Swedish PD case‐control material. Using a two‐sided χ test, we found significant association of rs2737029 (P= 0.003; χ2 =9.07) and rs356204 (P=0.048; χ2 =3.91) with disease, strengthening the involvement of SNCA polymorphisms in sporadic PD. Stratification of the human postmortem brain material by genotype of the three investigated polymorphisms, did not indicate any influence of genotype on α‐synuclein protein levels when comparing PD with controls. Taken together, our findings demonstrate that the investigated Parkinson patients have markedly reduced levels of α‐synuclein in cerebellum, and that this reduction is general, rather then correlated to the investigated polymorphisms, although two of the polymorphisms also associated with disease in a Swedish material.—Westerlund, M., Belin, A. C., Anvret, A., Håkansson, A., Nissbrandt, H., Lind, C., Sydow, O., Olson, L., and Galter, D. Cerebellar α‐synuclein levels are decreased in Parkinsons disease and do not correlate with SNCA polymorphisms associated with disease in a Swedish material. FASEB J. 22, 3509–3514 (2008)

Association of a protective paraoxonase 1 (PON1) polymorphism in Parkinson's disease

Pesticide exposure has been suggested to increase the risk to develop Parkinsons disease (PD). The arylesterase paraoxonase 1 (PON1) is mainly expressed in the liver and hydrolyzes organophosphates such as pesticides. The polymorphism Leu54Met (rs854560) in PON1, impairing enzyme activity and leading to decreased PON1 expression levels, has been reported to be associated with Parkinsons disease (PD). PON1 is part of a cluster on chromosome 7q21.3 together with PON2 and PON3. We investigated the occurrence of four additional polymorphisms in PON1 and two in PON2 in a Swedish PD case-control material. We found a significant association (p=0.007) with a PON1 promoter polymorphism, rs854571. The minor allele was more common among controls than PD cases which suggest a protective effect. This is strengthened by the fact that rs854571 is in strong linkage disequilibrium with another PON1 promoter polymorphism, rs854572, reported to increase PON1 gene expression. Our findings support the hypothesis that PON1 is involved in the etiology of PD and that higher PON1 levels are reducing the risk for PD.

Possible Involvement of a Mitochondrial Translation Initiation Factor 3 Variant Causing Decreased mRNA Levels in Parkinson's Disease

Genes important for mitochondrial function have been implicated in Parkinsons disease (PD). Mitochondrial translation initiation factor 3 (MTIF3) is a nuclear encoded protein required for the initiation of complex formation on mitochondrial ribosomes. Dysfunction of MTIF3 may impair mitochondrial function and dopamine neurons appear to be particularly vulnerable to oxidative stress, which may relate to their degeneration in PD. An association was recently reported between the synonymous rs7669(C>T) in MTIF3 and PD in a German case-control material. We investigated rs7669 in a Swedish Parkinson case-control material. The study revealed no significant association of the individual genotypes or alleles with PD. When comparing the combined TT/CT-genotypes versus the CC-genotype, we observed a significant association (P = .0473) with PD. We also demonstrated that the TT-genotype causes a significant decrease in MTIF3 mRNA expression compared to the CC-genotype (P = .0163). Our findings support the hypothesis that MTIF3 may be involved in the etiology of PD.

Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease

The serine‐protease OMI/HTRA2, required for several cellular processes, including mitochondrial function, autophagy, chaperone activity, and apoptosis, has been implicated in the pathogenesis of both Alzheimers disease (AD) and Parkinsons disease (PD). Western blot quantification of OMI/HTRA2 in frontal cortex of patients with AD (n=10) and control subjects (n=10) in two separate materials indicated reduced processed (active, 35 kDa) OMI/HTRA2 levels, whereas unprocessed (50 kDa) enzyme levels were not significantly different between the groups. Interestingly, the specific protease activity of OMI/HTRA2 was found to be significantly increased in patients with AD (n=10) compared to matched control subjects (n=10) in frontal cortex in two separate materials. Comparison of OMI/HTRA2 mRNA levels in frontal cortex and hippocampus, two brain areas particularly affected by AD, indicated similar levels in patients with AD (n=10) and matched control subjects (n=10). In addition, we analyzed the occurrence of the OMI/HTRA2 variants A141S and G399S in Swedish case‐control materials for AD and PD and found a weak association of A141S with AD, but not with PD. In conclusion, our genetic, histological, and biochemical findings give further support to an involvement of OMI/HTRA2 in the pathology of AD; however, further studies are needed to clarify the role of this gene in neurodegeneration.—Westerlund, M., Behbahani, H., Gellhaar, S., Forsell, C., Carmine Belin, A., Anvret, A., Zettergren, A., Nissbrandt, H., Lind, C., Sydow, O., Graff, C., Olson, L., Ankarcrona, M., Galter, D. Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimers disease. FASEB J. 25, 1345–1352 (2011). www.fasebj.org

Genetic studies of the protein kinase AKT1 in Parkinson's disease.

The protein kinase AKT1 belongs to the Akt family and is a potent mediator of cell growth and survival and fully activated when phosphorylated. The AKT family has been found to be phosphorylated to a lesser extent in the dopaminergic cells of Parkinsons disease patients compared to control individuals, which might influence cell survival. Several publications support the implication of AKT1 in disorders of the dopaminergic system including bipolar disease and schizophrenia. In 2008 an association study performed in a Greek Parkinsons disease case-control material reported the identification of a protective AKT1 haplotype. Based on their work we have performed a replication study in a Swedish Parkinsons disease cohort. We genotyped the four single nucleotide polymorphims (SNPs): rs2494743, rs2498788, rs2494746 and rs1130214 in a case-control material consisting of 243 Parkinson patients and 315 controls. We did not find any associations with Parkinsons disease for either the individual SNPs or any of the haplotypes. In contrast to previously published results, our data do not support the hypothesis of genetic variants in AKT1 confering protection against Parkinsons disease.

Modeling Parkinson's disease genetics: Altered function of the dopamine system in Adh4 knockout mice

Class IV alcohol dehydrogenase (ADH4) efficiently reduces aldehydes produced during lipid peroxidation, and may thus serve to protect from toxic effects of aldehydes e.g. on neurons. We hypothesized that ADH4 dysfunction may increase risk for Parkinsons disease (PD) and previously reported association of an ADH4 allele with PD. We found that a promoter polymorphism in this allele induced a 25-30% reduction of transcriptional activity. Based on these findings, we have now investigated whether Adh4 homo- (Adh4-/-) or heterozygous (Adh4+/-) knockout mice display any dopamine system-related changes in behavior, biochemical parameters or olfaction compared to wild-type mice. The spontaneous locomotor activity was found to be similar in the three groups, whereas administration of d-amphetamine or apomorphine induced a significant increase in horizontal activity in the Adh4-/- mice compared to wild-type mice. We measured levels of monoamines and their metabolites in striatum, frontal cortex and substantia nigra and found increased levels of dopamine and DOPAC in substantia nigra of Adh4-/- mice. Investigation of olfactory function revealed a reduced sense of smell in Adh4-/- mice accompanied by alterations in dopamine metabolite levels in the olfactory bulb. Taken together, our results suggest that lack of Adh4 gene activity induces changes in the function of the dopamine system, findings which are compatible with a role of loss-of-function mutations in ADH4 as possible risk factors for PD.

Adh1 and Adh1/4 knockout mice as possible rodent models for presymptomatic Parkinson's disease

Alcohol dehydrogenases (ADH) catalyze the reversible metabolism of many types of alcohols and aldehydes to prevent the possible toxic accumulation of these compounds. ADHs are of interest in Parkinsons disease (PD) since these compounds can be harmful to dopamine (DA) neurons. Genetic variants in ADH1C and ADH4 have been found to associate with PD and lack of Adh4 gene activity in a mouse model has recently been reported to induce changes in the DA system. Adh1 knockout (Adh1-/-) and Adh1/4 double knockout (Adh1/4-/-) mice were investigated for possible changes in DA system related activity, biochemical parameters and olfactory function compared to wild-type (WT) mice. Locomotor activity was tested at ∼7 (adult) and >15 months of age to mimic the late onset of PD. Adh1-/- and Adh1/4-/- mice displayed a significantly higher spontaneous locomotor activity than WT littermates. Both apomorphine and d-amphetamine increased total distance activity in Adh1-/- mice at both age intervals and in Adh1/4-/- mice at 7 months of age compared to WT mice. No significant changes were found regarding olfactory function, however biochemical data showed decreased 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratios in the olfactory bulb and decreased homovanillic acid (HVA)/DA ratios in the olfactory bulb, frontal cortex and striatum of Adh1/4-/- mice compared to WT mice. Our results suggest that lack of Adh1 alone or Adh1 and Adh4 together lead to changes in DA system related behavior, and that these knockout mice might be possible rodent models to study presymptomatic PD.