Neurodegenerative diseases studied in human brain and rodent models

Parkinson’s and Alzheimer’s disease are the two most common neurodegenerative diseases world-wide, but are still little understood. Papers in this thesis examine some of the possible pathogenic mechanisms with the help of mouse models and analysis of human post-mortem tissue. Gene-based animal models have been developed to study pathological pathways during disease progression. Using mouse models with overexpression or ablation of disease-related genes we analyzed effects of pathogenic mutations on the function of the proteins. A prominent feature of patients carrying the G2019S mutation in leucine rich repeat kinase 2 (LRRK2) is a high load of Lewy Bodies, an intracellular protein accumulation highly enriched in α-synuclein. We investigated the interaction of LRRK2 and α-synuclein in transgenic mice and did not find an influence of LRRK2 expression on fibrillization of α-synuclein or dopamine neurons pathology, indicating separate pathways for the two genes causing PD. Using another genetic model with a conditional knockout of the mitochondrial transcription factor A in dopamine neurons, the MitoPark mouse, we detected reduced dopamine release and pacemaker activity in the substantia nigra several weeks before these mice develop motor dysfunction. The mRNA levels of the hyperpolarization-activated cyclic nucleotide gated channels 1-4 (HCN1-4), responsible for the pacemaking activity, were not altered in MitoPark mice, indicating that post-translational modifications occur early in the presymptomatic stages of Parkinson’s disease. To evaluate behavioral and cellular changes related to L-DOPA therapy we used older MitoPark mice, which model late stages of Parkinson’s disease. Chronic L-DOPA treatment normalized gait parameters but induced also progressing dyskinetic behavior in MitoPark mice. The treatment also caused a robust increase of TH mRNA expression in the striatum, as evidenced by RNA-Sequencing. The induction of TH in striatal neurons with an interneuronal phenotype was dependent on the degree of dopamine depletion and the L-DOPA dose. In disease-affected brain areas of patients with Parkinson’s or Alzheimer’s disease we found a significant increase in small-sized cells expressing the lysosomal enzyme myeloperoxidase. This finding supports involvement of a neuroinflammatory component in these diseases and encourages the research for anti-inflammatory treatments. Another protein implicated in the pathogenesis of neurodegeneration is the serine peptidase HTRA2. We detected altered enzyme activity and expression of HTRA2 in frontal cortex samples from Alzheimer’s disease patients. The association of a mutation in a HTRA2 allele with Alzheimer’s disease in our case-control material further supported a role of mitochondrial dysfunction in the pathology. Taken together, the studies presented in this thesis uncover changes in gene and protein expression in mouse and human samples, as well as behavioral changes in animal models of disease and will aid the development of better treatment options by increasing our knowledge of underlying pathological mechanisms

Neurodegenerative phenotypes in an A53T α-synuclein transgenic mouse model are independent of LRRK2

Mutations in the genes encoding LRRK2 and α-synuclein cause autosomal dominant forms of familial Parkinson's disease (PD). Fibrillar forms of α-synuclein are a major component of Lewy bodies, the intracytoplasmic proteinaceous inclusions that are a pathological hallmark of idiopathic and certain familial forms of PD. LRRK2 mutations cause late-onset familial PD with a clinical, neurochemical and, for the most part, neuropathological phenotype that is indistinguishable from idiopathic PD. Importantly, α-synuclein-positive Lewy bodies are the most common pathology identified in the brains of PD subjects harboring LRRK2 mutations. These observations may suggest that LRRK2 functions in a common pathway with α-synuclein to regulate its aggregation. To explore the potential pathophysiological interaction between LRRK2 and α-synuclein in vivo, we modulated LRRK2 expression in a well-established human A53T α-synuclein transgenic mouse model with transgene expression driven by the hindbrain-selective prion protein promoter. Deletion of LRRK2 or overexpression of human G2019S-LRRK2 has minimal impact on the lethal neurodegenerative phenotype that develops in A53T α-synuclein transgenic mice, including premature lethality, pre-symptomatic behavioral deficits and human α-synuclein or glial neuropathology. We also find that endogenous or human LRRK2 and A53T α-synuclein do not interact together to influence the number of nigrostriatal dopaminergic neurons. Taken together, our data suggest that α-synuclein-related pathology, which occurs predominantly in the hindbrain of this A53T α-synuclein mouse model, occurs largely independently from LRRK2 expression. These observations fail to provide support for a pathophysiological interaction of LRRK2 and α-synuclein in vivo, at least within neurons of the mouse hindbrai

Neurodegenerative phenotypes in an A53T-synuclein transgenic mouse model are independent of LRRK2

Mutations in the genes encoding LRRK2 and -synuclein cause autosomal dominant forms of familial Parkinsons disease (PD). Fibrillar forms of -synuclein are a major component of Lewy bodies, the intracytoplasmic proteinaceous inclusions that are a pathological hallmark of idiopathic and certain familial forms of PD. LRRK2 mutations cause late-onset familial PD with a clinical, neurochemical and, for the most part, neuropathological phenotype that is indistinguishable from idiopathic PD. Importantly, -synuclein-positive Lewy bodies are the most common pathology identified in the brains of PD subjects harboring LRRK2 mutations. These observations may suggest that LRRK2 functions in a common pathway with -synuclein to regulate its aggregation. To explore the potential pathophysiological interaction between LRRK2 and -synuclein in vivo, we modulated LRRK2 expression in a well-established human A53T -synuclein transgenic mouse model with transgene expression driven by the hindbrain-selective prion protein promoter. Deletion of LRRK2 or overexpression of human G2019S-LRRK2 has minimal impact on the lethal neurodegenerative phenotype that develops in A53T -synuclein transgenic mice, including premature lethality, pre-symptomatic behavioral deficits and human -synuclein or glial neuropathology. We also find that endogenous or human LRRK2 and A53T -synuclein do not interact together to influence the number of nigrostriatal dopaminergic neurons. Taken together, our data suggest that -synuclein-related pathology, which occurs predominantly in the hindbrain of this A53T -synuclein mouse model, occurs largely independently from LRRK2 expression. These observations fail to provide support for a pathophysiological interaction of LRRK2 and -synuclein in vivo, at least within neurons of the mouse hindbrain

Glucocerebrosidase variant T369M is not a risk factor for Parkinson's disease in Sweden.

INTRODUCTION: Genetic variants in the Beta-glucocerebrosidase gene (GBA1) is a known risk factor for Parkinson's disease. The GBA1 mutations L444P, N370S and many other have been shown to associate with the disease in populations with diverse background. Some GBA1 polymorphisms have a less pronounced effect, and their pathogenicity has been debated. We have previously found associations with L444P, N370S and E326K and Parkinson's disease in Sweden. METHOD: In this study we used pyrosequencing to genotype the T369M variant in a large Swedish cohort consisting of 1,131 patients with idiopathic Parkinson's disease, and 1,594 control subjects to evaluate the possibility of this variant conferring an increased risk for Parkinson's disease. RESULTS: The minor allele frequency was 2.15% in patients and 1.76% in controls. Statistical analysis showed that there was no significant difference in allele frequency between patients and control subjects, p-value 0.37, Odds Ratio 1.23 with a 95% confidence interval of 0.82-1.83. CONCLUSION: Our results suggest that T369M is not a risk factor for Parkinson's disease in the Swedish population.Funding agencies: Swedish Research Council, The Swedish Parkinson Foundation, The Swedish Brain Foundation, Swedish Brain Power and the Karolinska Institutet Foundation and Funds, PS is a Wallenberg Clinical Scholar</p

Conditional expression of Parkinson's disease-related R1441C LRRK2 in midbrain dopaminergic neurons of mice causes nuclear abnormalities without neurodegeneration

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant Parkinson's disease (PD). The clinical and neurochemical features of LRRK2-linked PD are similar to idiopathic disease although neuropathology is somewhat heterogeneous. Dominant mutations in LRRK2 precipitate neurodegeneration through a toxic gain-of-function mechanism which can be modeled in transgenic mice overexpressing human LRRK2 variants. A number of LRRK2 transgenic mouse models have been developed that display abnormalities in dopaminergic neurotransmission and alterations in tau metabolism yet without consistently inducing dopaminergic neurodegeneration. To directly explore the impact of mutant LRRK2 on the nigrostriatal dopaminergic pathway, we developed conditional transgenic mice that selectively express human R1441C LRRK2 in dopaminergic neurons from the endogenous murine ROSA26 promoter. The expression of R1441C LRRK2 does not induce the degeneration of substantia nigra dopaminergic neurons or striatal dopamine deficits in mice up to 2 years of age, and fails to precipitate abnormal protein inclusions containing alpha-synuclein, tau, ubiquitin or autophagy markers (LC3 and p62). Furthermore, mice expressing R1441C LRRK2 exhibit normal motor activity and olfactory function with increasing age. Intriguingly, the expression of R1441C LRRK2 induces age-dependent abnormalities of the nuclear envelope in nigral dopaminergic neurons including reduced nuclear circularity and increased invaginations of the nuclear envelope. In addition, R1441C LRRK2 mice display increased neurite complexity of cultured midbrain dopaminergic neurons. Collectively, these novel R1441C LRRK2 conditional transgenic mice reveal altered dopaminergic neuronal morphology with advancing age, and provide a useful tool for exploring the pathogenic mechanisms underlying the R1441C LRRK2 mutation in PD. (C) 2014 Elsevier Inc All rights reserved

Strong association between glucocerebrosidase mutations and Parkinsons disease in Sweden

Several genetic studies have demonstrated an association between mutations in glucocerebrosidase (GBA), originally implicated in Gauchers disease, and an increased risk of Parkinsons disease (PD). We have investigated the possible involvement of genetic GBA variations in PD in the Swedish population. Three GBA variants, E326K, N370S, and L444P were screened in the largest Swedish Parkinson cohort reported to date; 1625 cases and 2025 control individuals. We found a significant association with high effect size of the rare variant L444P with PD (odds ratio 8.17; 95% confidence interval: 2.51-26.23; p-value = 0.0020) and a significant association of the common variant E326K (odds ratio 1.60; 95% confidence interval: 1.16-2.22; p-value = 0.026). The rare variant N370S showed a trend for association. Most L444P carriers (68%) were found to reside in northern Sweden, which is consistent with a higher prevalence of Gauchers disease in this part of the country. Our findings support the role of GBA mutations as risk factors for PD and point to lysosomal dysfunction as a mechanism contributing to PD etiology. (C) 2016 The Author(s). Published by Elsevier Inc.Funding Agencies|Swedish Brain Power; Swedish Research Council [K2013-99X-22248-01-3]; Swedish Parkinson Foundation [613/13, 712/14]; Swedish Brain Foundation [FO2013-0213]; Ake Wibergs Stiftelse [756194137]; Karolinska Institutet Funds [2013fobi37223]; Karolinska DPA within the Swedish National Health Services (ALF); Neurology Department Karolinska University Hospital 100-year Fund; ERC Advanced Investigator Grant [322744]; Swedish Parkinson Academy; Umea University (Insamlingsstiftelsen); Bundy Academy, Sweden; Lions Research Foundation Skane; Elsa Schmitz Stiftelse; Skane University Hospital Foundations; Donations program, NEURO forbundet, Sweden</p

Impaired nigrostriatal function precedes behavioral deficits in a genetic mitochondrial model of Parkinson's disease

Parkinson's disease (PD) involves progressive loss of nigrostriatal dopamine (DA) neurons over an extended period of time. Mitochondrial damage may lead to PD, and neurotoxins affecting mitochondria are widely used to produce degeneration of the nigrostriatal circuitry. Deletion of the mitochondrial transcription factor A gene (Tfam) in C57BL6 mouse DA neurons leads to a slowly progressing parkinsonian phenotype in which motor impairment is first observed at ∼12 wk of age. l-DOPA treatment improves motor dysfunction in these “MitoPark” mice, but this declines when DA neuron loss is more complete. To investigate early neurobiological events potentially contributing to PD, we compared the neurochemical and electrophysiological properties of the nigrostriatal circuit in behaviorally asymptomatic 6- to 8-wk-old MitoPark mice and age-matched control littermates. Release, but not uptake of DA, was impaired in MitoPark mouse striatal brain slices, and nigral DA neurons lacked characteristic pacemaker activity compared with control mice. Also, hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel function was reduced in MitoPark DA neurons, although HCN messenger RNA was unchanged. This study demonstrates altered nigrostriatal function that precedes behavioral parkinsonian symptoms in this genetic PD model. A full understanding of these presymptomatic cellular properties may lead to more effective early treatments of PD.—Good, C. H., Hoffman, A. F., Hoffer, B. J., Chefer, V. I., Shippenberg, T. S., Bäckman, C. M., Larsson, N.-G., Olson, L., Gellhaar, S., Galter, D., Lupica, C. R. Impaired nigrostriatal function precedes behavioral deficits in a genetic mitochondrial model of Parkinson's disease