Cortical hyperexcitability in mouse models and patients with amyotrophic lateral sclerosis is linked to noradrenaline deficiency.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by the death of upper (UMN) and lower motor neurons (LMN) in the motor cortex, brainstem, and spinal cord. Despite decades of research, ALS remains incurable, challenging to diagnose, and of extremely rapid progression. A unifying feature of sporadic and familial forms of ALS is cortical hyperexcitability, which precedes symptom onset, negatively correlates with survival, and is sufficient to trigger neurodegeneration in rodents. Using electrocorticography in the Sod1G86R and FusΔNLS/+ ALS mouse models and standard electroencephalography recordings in patients with sporadic ALS, we demonstrate a deficit in theta-gamma phase-amplitude coupling (PAC) in ALS. In mice, PAC deficits started before symptom onset, and in patients, PAC deficits correlated with the rate of disease progression. Using mass spectrometry analyses of CNS neuropeptides, we identified a presymptomatic reduction of noradrenaline (NA) in the motor cortex of ALS mouse models, further validated by in vivo two-photon imaging in behaving SOD1G93A and FusΔNLS/+ mice, that revealed pronounced reduction of locomotion-associated NA release. NA deficits were also detected in postmortem tissues from patients with ALS, along with transcriptomic alterations of noradrenergic signaling pathways. Pharmacological ablation of noradrenergic neurons with DSP-4 reduced theta-gamma PAC in wild-type mice and administration of a synthetic precursor of NA augmented theta-gamma PAC in ALS mice. Our findings suggest theta-gamma PAC as means to assess and monitor cortical dysfunction in ALS and warrant further investigation of the NA system as a potential therapeutic target.

The cause of eyelid ptosis, orthostatic hypotension and exercise intolerance.

To provide more insight in the delay in diagnosis and expectation of treatment adapted for the paediatrician, the data were collected from patients described with dopamine beta-hydroxylase deficiency are evaluated. More insight in clinical features of dopamine beta-hydroxylase deficiency consisting mainly of eyelid ptosis, orthostatic hypotension, hypoglycaemia and exercise intolerance, explains the delay in diagnosis of this congenital disorder, although all symptoms some more concealed are present. An increasing experience by L-DOPS, a resurrection for the patient, allows recommendations for early treatment. An explanation for the delay in diagnosis is provided together with the advice for treatment.

Clinical presentation and long-term follow-up of dopamine beta hydroxylase deficiency.

Dopamine beta hydroxylase (DBH) deficiency is an extremely rare autosomal recessive disorder with severe orthostatic hypotension, that can be treated with L-threo-3,4-dihydroxyphenylserine (L-DOPS). We aimed to summarize clinical, biochemical, and genetic data of all world-wide reported patients with DBH-deficiency, and to present detailed new data on long-term follow-up of a relatively large Dutch cohort. We retrospectively describe 10 patients from a Dutch cohort and 15 additional patients from the literature. We identified 25 patients (15 females) from 20 families. Ten patients were diagnosed in the Netherlands. Duration of follow-up of Dutch patients ranged from 1 to 21 years (median 13 years). All patients had severe orthostatic hypotension. Severely decreased or absent (nor)epinephrine, and increased dopamine plasma concentrations were found in 24/25 patients. Impaired kidney function and anemia were present in all Dutch patients, hypomagnesaemia in 5 out of 10. Clinically, all patients responded very well to L-DOPS, with marked reduction of orthostatic complaints. However, orthostatic hypotension remained present, and kidney function, anemia, and hypomagnesaemia only partially improved. Plasma norepinephrine increased and became detectable, while epinephrine remained undetectable in most patients. We confirm the core clinical characteristics of DBH-deficiency and the pathognomonic profile of catecholamines in body fluids. Impaired renal function, anemia, and hypomagnesaemia can be part of the clinical presentation. The subjective response to L-DOPS treatment is excellent and sustained, although the neurotransmitter profile in plasma does not normalize completely. Furthermore, orthostatic hypotension as well as renal function, anemia, and hypomagnesaemia improve only partially.

Central deficiency of norepinephrine synthesis and norepinephrinergic neurotransmission contributes to seizure-induced respiratory arrest.

Sudden unexpected death in epilepsy (SUDEP) is the leading cause of mortality in patients with intractable epilepsy. However, the pathogenesis of SUDEP seems to be poorly understood. Our previous findings showed that the incidence of seizure-induced respiratory arrest (S-IRA) was markedly reduced by atomoxetine in a murine SUDEP model. Because the central norepinephrine α-1 receptor (NEα-1R) plays a vital role in regulating respiratory function, we hypothesized that the suppression of S-IRA by atomoxetine was mediated by NE/NEα-1R interactions that can be reversed by NEα-1R antagonism. We examined whether atomoxetine-mediated suppression of S-IRA evoked by either acoustic stimulation or pentylenetetrazole (PTZ) in DBA/1 mice can be reversed by intraperitoneal (IP) and intracerebroventricular (ICV) administration of prazosin, a selective antagonist of NEα-1R. The content and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme for NE synthesis, in the lower brainstem was measured by ELISA. Electroencephalograms (EEG) were obtained from using the PTZ-evoked SUDEP model. In our models, atomoxetine-mediated suppression of S-IRA evoked by either acoustic stimulation or PTZ was significantly reversed by low doses of IP and ICV prazosin. Neither repetitive acoustic stimulation nor S-IRA reduced TH levels in lower brainstem. However, the enzyme activity of TH levels in lower brainstem was significantly increased by mechanical ventilation with DBA/1 mice, which makes the dying DBA/1 mice suffering from S-IRA and SUDEP recover. EEG data showed that although the protective effect of atomoxetine was reversed by prazosin, neither drug suppressed EEG activity. These data suggest that deficient synthesis of NE and norepinephrinergic neurotransmission contributed to S-IRA and that the NEα-1R is a potential therapeutic target for the prevention of SUDEP.

Noradrenergic circuits in the forebrain control affective responses to novelty.

RATIONALE: In rodents, exposure to novel environments elicits initial anxiety-like behavior (neophobia) followed by intense exploration (neophilia) that gradually subsides as the environment becomes familiar. Thus, innate novelty-induced behaviors are useful indices of anxiety and motivation in animal models of psychiatric disease. Noradrenergic neurons are activated by novelty and implicated in exploratory and anxiety-like responses, but the role of norepinephrine (NE) in neophobia has not been clearly delineated. OBJECTIVE: We sought to define the role of central NE transmission in neophilic and neophobic behaviors. METHODS: We assessed dopamine ß-hydroxylase knockout (Dbh -/-) mice lacking NE and their NE-competent (Dbh +/-) littermate controls in neophilic (novelty-induced locomotion; NIL) and neophobic (novelty-suppressed feeding; NSF) behavioral tests with subsequent quantification of brain-wide c-fos induction. We complimented the gene knockout approach with pharmacological interventions. RESULTS: Dbh -/- mice exhibited blunted locomotor responses in the NIL task and completely lacked neophobia in the NSF test. Neophobia was rescued in Dbh -/- mice by acute pharmacological restoration of central NE with the synthetic precursor L-3,4-dihydroxyphenylserine (DOPS), and attenuated in control mice by the inhibitory α2-adrenergic autoreceptor agonist guanfacine. Following either NSF or NIL, Dbh -/- mice demonstrated reduced c-fos in the anterior cingulate cortex, medial septum, ventral hippocampus, bed nucleus of the stria terminalis, and basolateral amygdala. CONCLUSION: These findings indicate that central NE signaling is required for the expression of both neophilic and neophobic behaviors. Further, we describe a putative noradrenergic novelty network as a potential therapeutic target for treating anxiety and substance abuse disorders.

Congenital absence of norepinephrine due to CYB561 mutations.

OBJECTIVE: Cytochrome b561 (CYB561) generates ascorbic acid, a cofactor in the enzymatic conversion of dopamine to norepinephrine by dopamine ß-hydroxylase. We propose that the clinical relevance of this pathway can be revealed by characterizing the autonomic and biochemical characteristics of patients with CYB561 mutations. METHODS: We performed autonomic evaluations in 4 patients with lifelong orthostatic hypotension in whom CYB561 mutations were determined by genomic sequencing. RESULTS: Patients had disabling lifelong orthostatic hypotension (OH) and impaired blood pressure response to the Valsalva maneuver (VM), with exaggerated hypotension during phase 2 and lack of overshoot during phase 4. Heart rate ratios for sinus arrhythmia and the VM were normal. Plasma norepinephrine and metabolites were undetectable, and plasma dopamine and metabolites were normal. Droxidopa restored norepinephrine levels and improved OH. Patients 1 and 2 were sisters and homozygous for a nonsense mutation in exon 2, c.131G>A, p.Trp44 (Circ Res 2018). Their brother (patient 3) died at age 16 and his DNA was not available. Patient 4 was compound heterozygous; one allele had a missense mutation in exon 2, c157C>T, p.His.53Tyr, and the other had an exon 2 deletion. CONCLUSION: CYB561 deficiency is characterized by selective sympathetic noradrenergic failure with lifelong, disabling OH but with normal sympathetic cholinergic (sweating) and parasympathetic (heart rate regulation) functions. We report a novel case of CYB561 deficiency due to an exon 2 deletion in one allele and a missense mutation in the other. These patients highlight the critical role CYB561 plays in sympathetic function and cardiovascular regulation.

Catecholamine-Synthesizing Enzymes in Pheochromocytoma and Extraadrenal Paraganglioma.

In chromaffin cells, tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), dopamine ß-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) are mainly involved in catecholamine synthesis. In this study, we evaluated the association between the status of catecholamine-synthesizing enzymes and histopathological features of pheochromocytoma and extraadrenal paraganglioma with special emphasis upon their postoperative clinical behavior. Immunohistochemical evaluation of TH, DBH, AADC, PNMT, Ki 67, and S-100 was performed in 29 pheochromocytoma and 10 extraadrenal paraganglioma and one lymph node harboring metastatic pheochromocytoma. Among these cases, metastasis was subsequently developed in three cases. Urinary normetanephrine (U-NM) levels were significantly higher in clinical metastatic cases than non-metastatic ones. Ki 67 labeling index was significantly higher in both clinical metastatic cases and the Adrenal Gland Scaled Score (PASS) score of ≧ 4 cases than PASS < 4 cases, although this score was originally used in pheochromocytoma. H-score of AADC and DBH were significantly lower in PASS ≧ 4 cases than those with < 4 cases, and in the cases associated with intratumoral necrosis (n = 4), the presence of spindle shaped tumor cells (n = 4), and large nests of cells or diffuse growth (n = 5). Lower status of intratumoral AADC could be related to poor differentiation of tumor cells in both catecholamine production and morphology and could be related to aggressive biological behavior of both pheochromocytoma and extraadrenal paraganglioma.

Localization of endogenous amyloid-ß to the coeruleo-cortical pathway: consequences of noradrenergic depletion.

The locus coeruleus (LC)-norepinephrine (NE) system is an understudied circuit in the context of Alzheimer's disease (AD), and is thought to play an important role in neurodegenerative and neuropsychiatric diseases involving catecholamine neurotransmitters. Understanding the expression and distribution of the amyloid beta (Aß) peptide, a primary component of AD, under basal conditions and under conditions of NE perturbation within the coeruleo-cortical pathway may be important for understanding its putative role in pathological states. Thus, the goal of this study is to define expression levels and the subcellular distribution of endogenous Aß with respect to noradrenergic profiles in the rodent LC and medial prefrontal cortex (mPFC) and, further, to determine the functional relevance of NE in modulating endogenous Aß42 levels. We report that endogenous Aß42 is localized to tyrosine hydroxylase (TH) immunoreactive somatodendritic profiles of the LC and dopamine-ß-hydroxylase (DßH) immunoreactive axon terminals of the infralimbic mPFC (ILmPFC). Male and female naïve rats have similar levels of amyloid precursor protein (APP) cleavage products demonstrated by western blot, as well as similar levels of endogenous Aß42 as determined by enzyme-linked immunosorbent assay. Two models of NE depletion, DSP-4 lesion and DßH knockout (KO) mice, were used to assess the functional relevance of NE on endogenous Aß42 levels. DSP-4 lesioned rats and DßH-KO mice show significantly lower levels of endogenous Aß42. Noradrenergic depletion did not change APP-cleavage products resulting from ß-secretase processing. Thus, resultant decreases in endogenous Aß42 may be due to decreased neuronal activity of noradrenergic neurons, or, by decreased stimulation of adrenergic receptors which are known to contribute to Aß42 production by enhancing γ-secretase processing under normal physiological conditions.

Hyperinsulinemia and Insulin Resistance in Dopamine ß-Hydroxylase Deficiency.

Context: Dopamine ß-hydroxylase (DBH) deficiency is a rare genetic disorder characterized by failure to convert dopamine to norepinephrine. DBH-deficient patients lack sympathetic adrenergic function and are therefore predisposed to orthostatic hypotension. DBH-deficient mice exhibit hyperinsulinemia, lower plasma glucose levels, and insulin resistance due to loss of tonic sympathetic inhibition of insulin secretion. The impact of DBH deficiency on glucose homeostasis in humans is unknown. Case Description: We describe the metabolic profile of an adolescent female DBH-deficient patient. The patient underwent genetic testing, cardiovascular autonomic function testing, and evaluation of insulin secretion and sensitivity with hyperglycemic clamp under treatment-naive conditions. All procedures were repeated after 1 year of treatment with the norepinephrine prodrug droxidopa (300 mg, 3 times a day). Genetic testing showed a homozygous mutation in the DBH gene (rs74853476). Under treatment-naive conditions, she had undetectable plasma epinephrine and norepinephrine levels, resulting in sympathetic noradrenergic failure and orthostatic hypotension (-32 mm Hg supine to seated). She had high adiposity (41%) and fasting plasma insulin levels (25 µU/mL), with normal glucose (91 mg/dL). Hyperglycemic clamp revealed increased glucose-stimulated insulin secretion and insulin resistance. Droxidopa restored plasma norepinephrine and improved orthostatic tolerance, with modest effects on glucose homeostasis. Conclusions: We provide evidence for impairment in cardiovascular autonomic regulation, hyperinsulinemia, enhanced glucose-stimulated insulin secretion, and insulin resistance in a DBH-deficient patient. These metabolic derangements were not corrected by chronic droxidopa treatment. These findings provide insight into the pathophysiology and treatment of DBH deficiency and into the importance of catecholaminergic mechanisms to resting metabolism.

Selective noradrenaline depletion impairs working memory and hippocampal neurogenesis.

Noradrenergic neurons in the locus coeruleus play a role in learning and memory, and their loss is an early event in Alzheimer's disease pathogenesis. Moreover, noradrenaline may sustain hippocampal neurogenesis; however, whether are these events related is still unknown. Four to five weeks following the selective immunotoxic ablation of locus coeruleus neurons, young adult rats underwent reference and working memory tests, followed by postmortem quantitative morphological analyses to assess the extent of the lesion, as well as the effects on proliferation and/or survival of neural progenitors in the hippocampus. When tested in the Water Maze task, lesioned animals exhibited no reference memory deficit, whereas working memory abilities were seen significantly impaired, as compared with intact or sham-lesioned controls. Stereological analyses confirmed a dramatic noradrenergic neuron loss associated to reduced proliferation, but not survival or differentiation, of 5-bromo-2'deoxyuridine-positive progenitors in the dentate gyrus. Thus, ascending noradrenergic afferents may be involved in more complex aspects of cognitive performance (i.e., working memory) possibly via newly generated progenitors in the hippocampus.

Skin biopsy and microneurography disclose selective noradrenergic dysfunction due to dopamine-ß-hydroxylase deficiency.

Skin biopsy and microneurography are autonomic tests directly evaluating adrenergic and cholinergic sympathetic fibers to identify selective deficiency of a specific peripheral sympathetic subdivision. We describe a patient with tomacular neuropathy due to a deletion of the PMP22 gene who complained of chronic orthostatic hypotension due to a dopamine-ß-hydroxylase deficiency confirmed by genetic analysis demonstrating two novel mutations in the DßH gene. To further characterize autonomic dysfunctions the proband underwent skin biopsy and microneurography. These tests disclosed a selective peripheral adrenergic dysfunction demonstrating the possibility to ascertain DßH deficiency. In conclusion, skin biopsy and microneurography may help to increase the diagnosis of this peculiar disorder particularly when routine autonomic nervous system tests show uncertain results.

Neurogenic orthostatic hypotension: roles of norepinephrine deficiency in its causes, its treatment, and future research directions.

BACKGROUND: Although a diversity of neurotransmitters and hormones participate in controlling blood pressure, norepinephrine released from postganglionic sympathetic nerve terminals is an important mediator of the rapid regulation of cardiovascular function required for homeostasis of cerebral perfusion. Hence, neurogenic orthostatic hypotension (NOH) often represents a deficiency of noradrenergic responsiveness to postural change. RESEARCH DESIGN AND METHODS: PubMed searches with 'orthostatic hypotension' and 'norepinephrine' as conjoint search terms and no restriction on language or date, so as to survey the pathophysiologic and clinical relevance of norepinephrine deficiency for current NOH interventions and for future directions in treatment and research. RESULTS: Norepinephrine deficiency in NOH can arise peripherally, due to cardiovascular sympathetic denervation (as in pure autonomic failure, Parkinson's disease, and a variety of neuropathies), or centrally, due to a failure of viscerosensory signals to generate adequate sympathetic traffic to intact sympathetic nerve endings (as in multiple system atrophy). Nonpharmacologic countermeasures such as pre-emptive water intake may yield blood-pressure increases exceeding those achieved pharmacologically. For patients with symptomatic NOH unresponsive to such strategies, a variety of pharmacologic interventions have been administered off-label on the basis of drug mechanisms expected to increase blood pressure via blood-volume expansion or vasoconstriction. Two pressor agents have received FDA approval: the sympathomimetic midodrine and more recently the norepinephrine prodrug droxidopa. CONCLUSIONS: Pressor agents are important for treating symptomatic NOH in patients unresponsive to lifestyle changes alone. However, the dysautonomia underlying NOH often permits blood-pressure excursions toward both hypotension and hypertension. Future research should aim to shed light on the resulting management issues, and should also explore the possibility of pharmacotherapy selectively targeting orthostatic blood-pressure decreases.

Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish.

Pharmacological studies in mammals suggest that norepinephrine (NE) plays an important role in promoting arousal. However, the role of endogenous NE is unclear, with contradicting reports concerning the sleep phenotypes of mice lacking NE due to mutation of dopamine ß-hydroxylase (dbh). To investigate NE function in an alternative vertebrate model, we generated dbh mutant zebrafish. In contrast to mice, these animals exhibit dramatically increased sleep. Surprisingly, despite an increase in sleep, dbh mutant zebrafish have a reduced arousal threshold. These phenotypes are also observed in zebrafish treated with small molecules that inhibit NE signaling, suggesting that they are caused by the lack of NE. Using genetic overexpression of hypocretin (Hcrt) and optogenetic activation of hcrt-expressing neurons, we also find that NE is important for Hcrt-induced arousal. These results establish a role for endogenous NE in promoting arousal and indicate that NE is a critical downstream effector of Hcrt neurons.

A patient with PMP22-related hereditary neuropathy and DBH-gene-related dysautonomia.

Recurrent focal neuropathy with liability to pressure palsies is a relatively frequent autosomal-dominant demyelinating neuropathy linked to peripheral myelin protein 22 (PMP22) gene deletions. The combination of PMP22 gene mutations with other genetic variants is known to cause a more severe phenotype than expected. We present the case of a patient with severe orthostatic hypotension since 12 years of age, who inherited a PMP22 gene deletion from his father. Genetic double trouble was suspected because of selective sympathetic autonomic disturbances. Through exome-sequencing analysis, we identified two novel mutations in the dopamine beta hydroxylase gene. Moreover, with interactome analysis, we excluded a further influence on the origin of the disease by variants in other genes. This case increases the number of unique patients presenting with dopamine-ß-hydroxylase deficiency and of cases with genetically proven double trouble. Finding the right, complete diagnosis is crucial to obtain adequate medical care and appropriate genetic counseling.

Aging-associated formaldehyde-induced norepinephrine deficiency contributes to age-related memory decline.

A norepinephrine (NE) deficiency has been observed in aged rats and in patients with Alzheimer's disease and is thought to cause cognitive disorder. Which endogenous factor induces NE depletion, however, is largely unknown. In this study, we investigated the effects of aging-associated formaldehyde (FA) on the inactivation of NE in vitro and in vivo, and on memory behaviors in rodents. The results showed that age-related DNA demethylation led to hippocampal FA accumulation, and when this occurred, the hippocampal NE content was reduced in healthy male rats of different ages. Furthermore, biochemical analysis revealed that FA rapidly inactivated NE in vitro and that an intrahippocampal injection of FA markedly reduced hippocampal NE levels in healthy adult rats. Unexpectedly, an injection of FA (at a pathological level) or 6-hydroxydopamine (6-OHDA, a NE depletor) can mimic age-related NE deficiency, long-term potentiation (LTP) impairments, and spatial memory deficits in healthy adult rats. Conversely, an injection of NE reversed age-related deficits in both LTP and memory in aged rats. In agreement with the above results, the senescence-accelerated prone 8 (SAMP8) mice also exhibited a severe deficit in LTP and memory associated with a more severe NE deficiency and FA accumulation, when compared with the age-matched, senescence-resistant 1 (SAMR1) mice. Injection of resveratrol (a natural FA scavenger) or NE into SAMP8 mice reversed FA accumulation and NE deficiency and restored the magnitude of LTP and memory. Collectively, these findings suggest that accumulated FA is a critical endogenous factor for aging-associated NE depletion and cognitive decline.