This work is a revised edition of the former article “Die Kalkulation irreparabler Mutationen” by the same author. New calculations have been included, and some unclear formulations have been eliminated. New is in the present edition above all the calculation of the very certain temperature - alterations which are necessary for the lengthening of monotonous sequence for one position, provided that these are responsible for a constant viscosity - change of the DNA surroundings (section 6, equations 94 and 95).

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are a major cause of Parkinson’s disease (PD), which is why modelling PD by replicating effects in animal models attracts great interest. However, the exact mechanisms of pathogenesis are still unclear. While a gain-of-function hypothesis generally receives consensus, there is evidence supporting an alternative loss-of-function explanation. Yet, neither overexpression of the human wild-type LRRK2 protein or its pathogenic variants, nor Lrrk2 knockout recapitulates key aspects of human PD in rodent models. Furthermore, there is conflicting evidence from morpholino knockdown studies in zebrafish regarding the extent of zygotic developmental abnormalities. Because reliable null mutants may be useful to infer gene function, and because the zebrafish is a more tractable laboratory vertebrate system than rodents to study disease mechanisms in vivo, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genomic editing was used to delete the ~60-kbp-long zebrafish lrrk2 locus containing the entire open reading frame. Constitutive removal of both the maternal and the zygotic lrrk2 function (mzLrrk2 individuals) causes a pleomorphic phenotype in the larval brain at 5 days post-fertilisation (dpf), including increased cell death, delayed myelination, and reduced and morphologically abnormal microglia/leukocytes. However, the phenotype is transient, spontaneously attenuating or resolving by 10 dpf, and the mutants are viable and fertile as adults. These observations are mirrored by whole-larva transcriptome data, revealing a more than eighteen-fold drop in the number of differentially expressed genes in mzLrrk2 larvae from 5 to 10 dpf. Additionally, analysis of spontaneous swimming activity shows hypokinesia as a predictor of Lrrk2 protein deficiency in larvae, but not in adult fish. Because the catecholaminergic (CA) neurons are the main clinically relevant target of PD in humans, the CA system of larvae and adult fish was analysed on both cellular and metabolic level. Despite an initial developmental delay at 5 dpf, the CA system is structurally intact at 10 dpf and later on in adult fish aged 6 and 11 months. However, monoamine oxidase (Mao)-dependent degradation of biogenic amines, including dopamine, is increased in older fish, possibly suggesting impaired synaptic transmission or a leading cause of cell damage in the long term. Furthermore, decreased mitosis rate in the larval brain was found, in the anterior portion only at 5 dpf, strongly and throughout the whole organ at 10 dpf. Conceivably, lrrk2 may have a more general role in the control of cell proliferation during early development and a more specialised one in the adult stage, possibly conditional, for example upon brain damage. Because the zebrafish can regenerate lost neurons, it represents a unique opportunity to elucidate the endogenous processes that may counteract neurodegeneration in a predisposing genetic background. To this aim, the regenerative potential of the adult telencephalon upon stab injury was tested in mzLrrk2 fish. Indeed, neuronal proliferation was reduced, suggesting that a complete understanding of Lrrk2 biology may not be fully appreciated without recreating challenging scenarios. To summarise, the present results demonstrate that loss of lrrk2 has an early effect on zebrafish brain development that is later often compensated. Nonetheless, perturbed aminergic catabolism, and specifically increased Mao-dependent aminergic degradation, is reported for the first time in a LRRK2 knockout model. Furthermore, a link between Lrrk2 and the control of basal cell proliferation in the brain, which may become critical under challenging circumstances such as brain injury, is proposed. Future directions should aim at exploring which brain cell types are specifically affected by the mzLrrk2 hypoproliferative phenotype and the resulting consequences on a circuitry level, particularly in very old fish (i.e., over 2 years of age).

Bei der Replikation monotoner Sequenzen tritt theoretisch ein Vorgang auf, den wir als „Basenkonkurrenz“ bezeichnen: Da sich an jeder Replikations-Stelle mehrere Basenbausteine bewerben, aber immer nur einer benötigt wird, bewerben sich die übrig gebliebenen Bausteine an den jeweils nächsten Replikations - Positionen und erlangen wegen der fortwährenden Beschleunigung durch elektrostatische Anziehung immer größere kinetische Energien. Das führt dazu, dass an einer bestimmten Stelle der replizierenden monotonen Sequenz der eine Partner der Wasserstoffbrückenbindung ein hohes Energieniveau erreicht. Es wird berechnet, dass sich dadurch kurzzeitig eine sehr hohe Bindungsenergie zwischen den beiden Partnern der Wasserstoffbrückenbindung einstellt, wodurch der in dieser kurzen Zeitspanne wirkende DNA-Reparaturmechanismus unterdrückt wird. Die Auswirkungen der hohen Basenkonkurrenz – Energien werden berechnet (hohe Bindungsenergien der Wasserstoffbrückenbindungen, Tunnelvorgänge, irreparable Mutationen). Die Folgen dieser Erscheinung sind Tumorbildung, Alterung, Veränderung der DNA – Struktur, Beeinflussung der Evolution, worauf im Einzelnen eingegangen wird. Es zeigt sich, dass die negativen Auswirkungen der Basenkonkurrenz vorwiegend bei zu niedriger Viskosität des Zellplasmas auftreten.