Human Bacterial Artificial Chromosome (BAC) Transgenesis Fully Rescues Noradrenergic Function in Dopamine β-Hydroxylase Knockout Mice

Abstract

Dopamine β-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE) in noradrenergic/adrenergic cells. DBH deficiency prevents NE production and causes sympathetic failure, hypotension and ptosis in humans and mice; DBH knockout (Dbh -/-) mice reveal other NE deficiency phenotypes including embryonic lethality, delayed growth, and behavioral defects. Furthermore, a single nucleotide polymorphism (SNP) in the human DBH gene promoter (-970C>T; rs1611115) is associated with variation in serum DBH activity and with several neurological- and neuropsychiatric-related disorders, although its impact on DBH expression is controversial. Phenotypes associated with DBH deficiency are typically treated with L-3,4-dihydroxyphenylserine (DOPS), which can be converted to NE by aromatic acid decarboxylase (AADC) in the absence of DBH. In this study, we generated transgenic mice carrying a human bacterial artificial chromosome (BAC) encompassing the DBH coding locus as well as ~45 kb of upstream and ~107 kb of downstream sequence to address two issues. First, we characterized the neuroanatomical, neurochemical, physiological, and behavioral transgenic rescue of DBH deficiency by crossing the BAC onto a Dbh -/- background. Second, we compared human DBH mRNA abundance between transgenic lines carrying either a “C” or a “T” at position -970. The BAC transgene drove human DBH mRNA expression in a pattern indistinguishable from the endogenous gene, restored normal catecholamine levels to the peripheral organs and brain of Dbh -/- mice, and fully rescued embryonic lethality, delayed growth, ptosis, reduced exploratory activity, and seizure susceptibility. In some cases, transgenic rescue was superior to DOPS. However, allelic variation at the rs1611115 SNP had no impact on mRNA levels in any tissue. These results indicate that the human BAC contains all of the genetic information required for tissue-specific, functional expression of DBH and can rescue all measured Dbh deficiency phenotypes, but did not reveal an impact of the rs11115 variant on DBH expression in mice.

Noradrenergic circuits in the forebrain control affective responses to novelty

“…We found that over the course of 1 h, NE-deficient mice exhibited reduced locomotor activity and more rapid habituation in the novel test environment compared to control mice. These findings are congruent with previous reports from our lab and suggest that novelty may have reduced incentive value or intrinsic salience in NE-deficient animals (Cubells et al 2016;Porter-Stransky et al 2019;Weinshenker et al 2002a). Importantly, novel environments elicit an initial increase in locomotion in Dbh -/-mice that habituates over time, suggesting that spatial memory mechanisms that distinguish novel and familiar environments remain intact in the absence of NE.…”

“…In the NIL test, high locomotor reactivity to novelty is interpreted as increased exploratory behavior, while low locomotor responses are interpreted as decreased exploratory behavior (Cubells et al 2016;Schroeder et al 2013;Stone et al 1999). Neophobia can be assessed in mice with the novelty-suppressed feeding (NSF) test, a conflict-based model of anxiety-like behavior in which anxiety elicited by exposure to an unfamiliar environment suppresses feeding in hungry mice presented with food (Cryan and Sweeney 2011;Dulawa et al 2005).…”

GPR37L1 modulates seizure susceptibility: Evidence from mouse studies and analyses of a human GPR37L1 variant

“…Each mouse was placed in a clear, Plexiglas chamber contained within a chemical fume hood, and flurothyl was introduced into the chamber (20 μl/min) such that it vaporized and filled the chamber. Latencies to the first myoclonic jerk and generalized tonic-clonic seizure (GTCS) were recorded 33; 34 Mice were removed from the chamber immediately after the GTCS.…”

Down but Not Out: The Consequences of Pretangle Tau in the Locus Coeruleus

“…Exclusive anatomic targeting of tau fibrils to the LC is technically very challenging due to the small size of this nucleus (1500 neurons in a rodent), and there is no way to ensure noradrenergic neuron specificity. By contrast, genetic strategies have been successful in targeting LC neurons to produce effector and reporter proteins with cell-type specificity through promoter-driven expression using NE synthesis enzymes (TH, DBH) [ 81 – 83 ], LC transcription factors (Phox2b) [ 84 , 85 ], and LC specification genes (Ear2 and DBH/En1) [ 36 , 86 , 87 ]. Recent work has shown selective and robust viral-mediated expression using Phox2b-dependent promoters [ 84 , 85 ], Cre-dependent expression in TH-Cre mice [ 81 , 88 ], or microinjections of virus containing a constitutive promoter [ 89 ].…”