Riddles in the dark: Decoding the relationship between neuromelanin and neurodegeneration in locus coeruleus neurons.

The noradrenergic locus coeruleus (LC) is among the first regions of the brain affected by pathology in both Alzheimer's disease (AD) and Parkinson's disease (PD), but the reasons for this selective vulnerability are not completely understood. Several features of LC neurons have been proposed as contributing factors to this dysfunction and degeneration, and this review will focus on the presence of neuromelanin (NM). NM is a dark pigment unique to catecholaminergic cells that is formed of norepinephrine (NE) and dopamine (DA) metabolites, heavy metals, protein aggregates, and oxidated lipids. We cover what is currently known about NM and the limitations of historical approaches, then discuss the new human tyrosinase (hTyr) model of NM production in rodent catecholamine cells in vivo that offers unique opportunities for studying its neurobiology, neurotoxicity, and potential of NM-based therapeutics for treating neurodegenerative disease.

Tyrosinase-induced neuromelanin accumulation triggers rapid dysregulation and degeneration of the mouse locus coeruleus.

Motor symptoms in Parkinson's disease (PD) are caused by degeneration of dopamine (DA) neurons of the substantia nigra (SN), while early non-motor symptoms such as anxiety and sleep disturbances are likely mediated by dysfunction of locus coeruleus (LC) norepinephrine (NE) neurons. The LC develops α-synuclein pathology prior to SN DA neurons in PD, and later undergoes degeneration, but the mechanisms responsible for its vulnerability are unknown. The SN and LC are the only structures in the brain that produces appreciable amounts of neuromelanin (NM), a dark cytoplasmic pigment. It has been proposed that NM initially plays a protective role by sequestering toxic catecholamine metabolites and heavy metals, but may become harmful during aging and PD as they overwhelm cellular machinery and are released during neurodegeneration. Rodents do not naturally produce NM, limiting the study of causal relationships between NM and PD-associated LC pathology. Adapting a viral-mediated approach for expression of human tyrosinase, the enzyme responsible for peripheral melanin production, we successfully promoted pigmentation in mouse LC neurons that recapitulates key features of endogenous NM found in primates, including eumelanin and pheomelanin, lipid droplets, and a double-membrane encasement. Pigment expression results in mild neurodegeneration, reduced NE levels, transcriptional changes, and novelty-induced anxiety phenotypes as early as 1-week post-injection. By 6-weeks, NM accumulation is associated with severe LC neurodegeneration and a robust neuroinflammatory response. These phenotypes are reminiscent of LC dysfunction in PD, validating this model for studying the consequences of pigment accumulation in the LC as it relates to neurodegenerative disease.

The Neurotoxin DSP-4 Dysregulates the Locus Coeruleus-Norepinephrine System and Recapitulates Molecular and Behavioral Aspects of Prodromal Neurodegenerative Disease.

The noradrenergic locus coeruleus (LC) is among the earliest sites of tau and α-synuclein pathology in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. The onset of these pathologies coincides with loss of noradrenergic fibers in LC target regions and the emergence of prodromal symptoms including sleep disturbances and anxiety. Paradoxically, these prodromal symptoms are indicative of a noradrenergic hyperactivity phenotype, rather than the predicted loss of norepinephrine (NE) transmission following LC damage, suggesting the engagement of complex compensatory mechanisms. Because current therapeutic efforts are targeting early disease, interest in the LC has grown, and it is critical to identify the links between pathology and dysfunction. We employed the LC-specific neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), which preferentially damages LC axons, to model early changes in the LC-NE system pertinent to AD and PD in male and female mice. DSP-4 (two doses of 50 mg/kg, one week apart) induced LC axon degeneration, triggered neuroinflammation and oxidative stress, and reduced tissue NE levels. There was no LC cell death or changes to LC firing, but transcriptomics revealed reduced expression of genes that define noradrenergic identity and other changes relevant to neurodegenerative disease. Despite the dramatic loss of LC fibers, NE turnover and signaling were elevated in terminal regions and were associated with anxiogenic phenotypes in multiple behavioral tests. These results represent a comprehensive analysis of how the LC-NE system responds to axon/terminal damage reminiscent of early AD and PD at the molecular, cellular, systems, and behavioral levels, and provides potential mechanisms underlying prodromal neuropsychiatric symptoms.

Norepinephrine and dopamine contribute to distinct repetitive behaviors induced by novel odorant stress in male and female mice.

Exposure to unfamiliar odorants induces an array of repetitive defensive and non-defensive behaviors in rodents which likely reflect adaptive stress responses to the uncertain valence of novel stimuli. Mice genetically deficient for dopamine ß-hydroxylase (Dbh-/-) lack the enzyme required to convert dopamine (DA) into norepinephrine (NE), resulting in globally undetectable NE and supranormal DA levels. Because catecholamines modulate novelty detection and reactivity, we investigated the effects of novel plant-derived odorants on repetitive behaviors in Dbh-/- mice and Dbh+/- littermate controls, which have catecholamine levels comparable to wild-type mice. Unlike Dbh+/- controls, which exhibited vigorous digging in response to novel odorants, Dbh-/- mice displayed excessive grooming. Drugs that block NE synthesis or neurotransmission suppressed odorant-induced digging in Dbh+/- mice, while a DA receptor antagonist attenuated grooming in Dbh-/- mice. The testing paradigm elicited high circulating levels of corticosterone regardless of Dbh genotype, indicating that NE is dispensable for this systemic stress response. Odorant exposure increased NE and DA abundance in the prefrontal cortex (PFC) of Dbh+/- mice, while Dbh-/- animals lacked NE and had elevated PFC DA levels that were unaffected by novel smells. Together, these findings suggest that novel odorant-induced increases in central NE tone contribute to repetitive digging and reflect psychological stress, while central DA signaling contributes to repetitive grooming. Further, we have established a simple method for repeated assessment of stress-induced repetitive behaviors in mice, which may be relevant for modeling neuropsychiatric disorders like Tourette syndrome or obsessive-compulsive disorder that are characterized by stress-induced exacerbation of compulsive symptoms.

Transgenic Mice Expressing Human α-Synuclein in Noradrenergic Neurons Develop Locus Ceruleus Pathology and Nonmotor Features of Parkinson's Disease.

Degeneration of locus ceruleus (LC) neurons and dysregulation of noradrenergic signaling are ubiquitous features of Parkinson's disease (PD). The LC is among the first brain regions affected by α-synuclein (asyn) pathology, yet how asyn affects these neurons remains unclear. LC-derived norepinephrine (NE) can stimulate neuroprotective mechanisms and modulate immune cells, while dysregulation of NE neurotransmission may exacerbate disease progression, particularly nonmotor symptoms, and contribute to the chronic neuroinflammation associated with PD pathology. Although transgenic mice overexpressing asyn have previously been developed, transgene expression is usually driven by pan-neuronal promoters and thus has not been selectively targeted to LC neurons. Here we report a novel transgenic mouse expressing human wild-type asyn under control of the noradrenergic-specific dopamine ß-hydroxylase promoter (DBH-hSNCA). These mice developed oligomeric and conformation-specific asyn in LC neurons, alterations in hippocampal and LC microglial abundance, upregulated GFAP expression, degeneration of LC fibers, decreased striatal DA metabolism, and age-dependent behaviors reminiscent of nonmotor symptoms of PD that were rescued by adrenergic receptor antagonists. These mice provide novel insights into how asyn pathology affects LC neurons and how central noradrenergic dysfunction may contribute to early PD pathophysiology.SIGNIFICANCE STATEMENT ɑ-Synuclein (asyn) pathology and loss of neurons in the locus ceruleus (LC) are two of the most ubiquitous neuropathologic features of Parkinson's disease (PD). Dysregulated norepinephrine (NE) neurotransmission is associated with the nonmotor symptoms of PD, including sleep disturbances, emotional changes such as anxiety and depression, and cognitive decline. Importantly, the loss of central NE may contribute to the chronic inflammation in, and progression of, PD. We have generated a novel transgenic mouse expressing human asyn in LC neurons to investigate how increased asyn expression affects the function of the central noradrenergic transmission and associated behaviors. We report cytotoxic effects of oligomeric and conformation-specific asyn, astrogliosis, LC fiber degeneration, disruptions in striatal dopamine metabolism, and age-dependent alterations in nonmotor behaviors without inclusions.

Central norepinephrine transmission is required for stress-induced repetitive behavior in two rodent models of obsessive-compulsive disorder.

RATIONALE: Obsessive-compulsive disorder (OCD) is characterized by repetitive behaviors exacerbated by stress. Many OCD patients do not respond to available pharmacotherapies, but neurosurgical ablation of the anterior cingulate cortex (ACC) can provide symptomatic relief. Although the ACC receives noradrenergic innervation and expresses adrenergic receptors (ARs), the involvement of norepinephrine (NE) in OCD has not been investigated. OBJECTIVE: To determine the effects of genetic or pharmacological disruption of NE neurotransmission on marble burying (MB) and nestlet shredding (NS), two animal models of OCD. METHODS: We assessed NE-deficient (Dbh -/-) mice and NE-competent (Dbh +/-) controls in MB and NS tasks. We also measured the effects of anti-adrenergic drugs on NS and MB in control mice and the effects of pharmacological restoration of central NE in Dbh -/- mice. Finally, we compared c-fos induction in the locus coeruleus (LC) and ACC of Dbh -/- and control mice following both tasks. RESULTS: Dbh -/- mice virtually lacked MB and NS behaviors seen in control mice but did not differ in the elevated zero maze (EZM) model of general anxiety-like behavior. Pharmacological restoration of central NE synthesis in Dbh -/- mice completely rescued NS behavior, while NS and MB were suppressed in control mice by anti-adrenergic drugs. Expression of c-fos in the ACC was attenuated in Dbh -/- mice after MB and NS. CONCLUSION: These findings support a role for NE transmission to the ACC in the expression of stress-induced compulsive behaviors and suggest further evaluation of anti-adrenergic drugs for OCD is warranted.