**3. Consequence of LC neuronal loss in PD**

#### **3.1 Alterations in terminal noradrenergic markers**

The loss of LC noradrenergic neurons in PD subjects should result in changes in NE levels at the various forebrain regions it innervates and the degree of loss should depend on the amount of LC innervation the region receives as compared to lateral tegmental groups. In PD subjects, there are reduced NE levels in the cortex, hypothalamus and cerebellum (Gasper et al., 1984, 1991; Kish et al., 1984; Shannak et al., 1994). TH- and DBHimmunoreactivity (IR) are also reduced in the cortex of PD subjects (Gasper et al., 1991). The reduction in neurotransmitter content and synthesizing enzymes in PD subjects can be attributed to the loss of noradrenergic innervation to these areas associated with the loss of LC noradrenergic neurons. The cortex, as indicated above, receives sole innervation from the LC, so changes in LC neuronal number would have a major impact in this region (Aston-Jones et al., 1995; Jones & Moore, 1977; Loughlin et al., 1986a, b; Mason & Fibiger, 1970; Moore & Bloom, 1979; Olsen & Fuxe, 1971; Ungerstedt, 1971; Waterhouse et al., 1983). Presently, the consequence of LC noradrenergic neuronal loss in PD on NE tissue content in the dopaminergic SN region is unknown.

#### **3.2 Alterations in noradrenergic markers in LC**

In contrast to terminal NE content in forebrain regions, NE levels in the LC of PD subjects does not appear to be different from controls (Cash et al., 1987). Examining different noradrenergic markers in the surviving LC neurons in PD subjects indicates the noradrenergic neurons are not compensating for the loss of surrounding neurons or terminal NE. The number of TH and DBH mRNA positively labeled neurons in the LC of PD subjects are significantly reduced as compared to age-matched control subjects (Szot, 2000, 2006; McMillan et al., 2011) and corresponds to the documented loss of noradrenergic neurons by other laboratories (Bertrand et al., 1997; Cash et al., 1987; Chan-Palay & Asan, 1989; Hornykiewicz & Kish, 1987; Marien et al., 2004; McMillan et al., 2011; Patt & Gerhard, 1993; Zarow et al., 2003). The degree of LC noradrenergic neuronal loss determined by TH and DBH mRNA expression in PD subjects is verified by counting the number of LC TH-IR positive labeled neurons (McMillan et al., 2011). TH mRNA expression/neuron of the

The Noradrenergic System is a Major Component in Parkinson's Disease 251

neurons (Huot & Parent, 2007; Yamada et al., 2007). Orthostatic hypotension, which is attributed to reduced CNS NE function, is relieved only with the administration of DOPS (Goldstein et al., 2011). DOPS is also used to normalize CNS levels of NE in the DBH knockout mouse that lacks the synthetic enzyme DBH that converts DA to NE (Figure 1) (Thomas et al., 1998). So these data would suggest that NE function in untreated PD and

Fig. 2. CSF catecholamine levels in PD subjects on chronic L-DOPA treatment and age-

**4. Comparison of the consequence of LC neuronal loss between PD and** 

noradrenergic neurons in AD may be compensating for the lost neurons.

Alzheimer's disease (AD), another neurodegenerative disorder, also exhibits significant LC noradrenergic neuronal loss (Bondareff et al., 1982; Chan-Palay & Asan, 1989; German et al., 1992; Mann et al., 1980; Marcyniuk et al., 1986; Szot et al., 2000, 2006; Tomlinson et al., 1981). However, unlike PD, in AD subjects the surviving LC noradrenergic neurons appear to be compensating for the loss. The content of NE in terminal regions of postmortem AD subjects is reduced, but the reduction does not correspond to the degree of neuronal loss (Adolfsson et al., 1979; Hoogendijk et al., 1999; Mann et al., 1981; Palmer et al., 1987; Reinikainen et al., 1988; Toghi et al., 1992; Tomlinson et al., 1981). Similar results were observed for IR of NE-synthesizing enzymes in the forebrain of postmortem AD subjects (Cross et al., 1981; Palmer et al., 1987; Perry et al., 1981; Russo-Neustadt et al., 1998). These data indicate that there is a reduction in NE function in forebrain regions of AD subjects, but it doesn't correlate to the degree of LC neuronal loss even in a region like the cortex which receives all of its innervation from the LC (Aston-Jones et al., 1995; Jones & Moore, 1977; Loughlin et al., 1986a, b; Mason & Fibiger, 1970; Moore & Bloom, 1979; Olsen & Fuxe, 1971; Ungerstedt, 1971; Waterhouse et al., 1983). These data suggest the surviving LC

approximately 4 hours after administration of L-DOPA. A) L-DOPA level, B) DA and NE levels and C) DOPAC and DHPG CSF levels in control and PD subjects on L-DOPA

treatment. Catecholamine levels were extracted by alumina extraction and analyzed by high performance liquid chromatography (HPLC) as previously described (Szot et al., 2010). Each point represents a single individual catecholamine value for control and PD subjects, with average and standard error means (SEM) illustrated in bars for each catecholamine analyzed. \* Indicates significant difference between control and PD subjects.

matched control subjects. CSF spinal fluid was taken at the same time of day,

possibly treated subjects is reduced.

**Alzheimer's disease (AD)** 

**4.1 Alterations in terminal noradrenergic markers** 

surviving LC noradrenergic neurons in PD subjects did not differ from TH mRNA expression/neuron in age-matched control subjects, indicating a lack of compensation of the surviving LC neurons (McMillan et al., 2011). Another marker of noradrenergic neurons, NE transporter (NET), was measured in the LC of age-matched and PD subjects. NET's function is to remove released NE from the synapse. Therefore, NETs are localized only to noradrenergic cell bodies, dendrites (peri-LC dendritic zone), and axon terminals of noradrenergic neurons. NET binding over cell body region and peri-LC dendritic zone in PD subjects is significantly reduced compared to age-matched control subjects and the loss of NET binding over the cell body region corresponds to the number of LC noradrenergic neurons in the LC (McMillan et al., 2011). The loss of NET binding in the peri-LC dendritic zone in PD subjects indicates the surviving LC neurons are not compensating for neuronal loss by increasing dendritic innervation. The loss of dendritic innervation in PD subjects, as determined by NET binding, is supported by reduced dendritic TH-IR labeling in the LC region of PD subjects as compared to age-matched controls (McMillan et al., 2011). However, the surviving LC neurons in PD subjects do demonstrate compensation in the expression of DBH mRNA (McMillan et al., 2011). The increased DBH mRNA expression/neuron in the surviving LC neurons of PD subjects may not be a response to neuronal loss because DBH mRNA expression/neuron is not altered in another neurodegenerative disorder with a significant loss of LC noradrenergic neurons (see below). Therefore, the increase in DBH mRNA expression/neuron observed in the surviving LC neurons in PD subjects may be a response to some other factor particular to PD, although it is unclear what that factor might be. The consequence of increased DBH mRNA expression of surviving LC neurons on NE levels in PD subjects is also unclear. Studies measuring cerebral spinal fluid (CSF) NE levels in PD subjects are variable. CSF NE levels and metabolites vary from reduced to no difference in postmortem and alive PD subjects (withdrawn from L-DOPA treatment) as compared to control subjects (Chia et al., 1993, 1995; Mann et al., 1983; Scatton et al., 1986; Turkka et al., 1987). As indicated above, NE tissue content is reduced, but LC concentration is similar to control subjects. These data suggest that NE levels and function may be reduced in PD, similar to DA levels and function.

#### **3.3 Effect of L-DOPA treatment on noradrenergic system**

The "standard gold" treatment for PD is L-DOPA. Administration of L-DOPA alleviates the symptoms associated with the loss of dopaminergic neurons (Cotzias et al., 1969); however, L-DOPA does not necessarily alleviate the non-motor symptoms associated with the loss of noradrenergic neurons (Sethi, 2008). Chronic administration of L-DOPA to PD subjects significantly elevates CSF levels of L-DOPA, DA and the DA metabolite dihydroxyphenylacetic acid (DOPAC) from age-matched control subjects (Figure 2A, B and C). There is a positive correlation of CSF DA levels to CSF L-DOPA levels. CSF NE levels in PD subjects on chronic L- DOPA treatment are not statistically different from age-matched controls (Figure 2B), but the NE metabolite 3,4-dihydroxyphenylglycol (DHPG) is significantly elevated in PD patients on chronic L-DOPA (Figure 2C). It is unclear why NE levels do not correlate to CSF L-DOPA levels in PD subjects like DA. A possible reason for the discrepancy in NE and DA levels in PD subjects on chronic L-DOPA therapy may be because administered L-DOPA is decarboxylated to DA in striatal neurons (as compared to DA from SN terminals) and serotonergic neurons, not in the few remaining dopaminergic

surviving LC noradrenergic neurons in PD subjects did not differ from TH mRNA expression/neuron in age-matched control subjects, indicating a lack of compensation of the surviving LC neurons (McMillan et al., 2011). Another marker of noradrenergic neurons, NE transporter (NET), was measured in the LC of age-matched and PD subjects. NET's function is to remove released NE from the synapse. Therefore, NETs are localized only to noradrenergic cell bodies, dendrites (peri-LC dendritic zone), and axon terminals of noradrenergic neurons. NET binding over cell body region and peri-LC dendritic zone in PD subjects is significantly reduced compared to age-matched control subjects and the loss of NET binding over the cell body region corresponds to the number of LC noradrenergic neurons in the LC (McMillan et al., 2011). The loss of NET binding in the peri-LC dendritic zone in PD subjects indicates the surviving LC neurons are not compensating for neuronal loss by increasing dendritic innervation. The loss of dendritic innervation in PD subjects, as determined by NET binding, is supported by reduced dendritic TH-IR labeling in the LC region of PD subjects as compared to age-matched controls (McMillan et al., 2011). However, the surviving LC neurons in PD subjects do demonstrate compensation in the expression of DBH mRNA (McMillan et al., 2011). The increased DBH mRNA expression/neuron in the surviving LC neurons of PD subjects may not be a response to neuronal loss because DBH mRNA expression/neuron is not altered in another neurodegenerative disorder with a significant loss of LC noradrenergic neurons (see below). Therefore, the increase in DBH mRNA expression/neuron observed in the surviving LC neurons in PD subjects may be a response to some other factor particular to PD, although it is unclear what that factor might be. The consequence of increased DBH mRNA expression of surviving LC neurons on NE levels in PD subjects is also unclear. Studies measuring cerebral spinal fluid (CSF) NE levels in PD subjects are variable. CSF NE levels and metabolites vary from reduced to no difference in postmortem and alive PD subjects (withdrawn from L-DOPA treatment) as compared to control subjects (Chia et al., 1993, 1995; Mann et al., 1983; Scatton et al., 1986; Turkka et al., 1987). As indicated above, NE tissue content is reduced, but LC concentration is similar to control subjects. These data suggest that NE levels and function may be reduced in PD, similar to DA levels and

function.

**3.3 Effect of L-DOPA treatment on noradrenergic system** 

The "standard gold" treatment for PD is L-DOPA. Administration of L-DOPA alleviates the symptoms associated with the loss of dopaminergic neurons (Cotzias et al., 1969); however, L-DOPA does not necessarily alleviate the non-motor symptoms associated with the loss of noradrenergic neurons (Sethi, 2008). Chronic administration of L-DOPA to PD subjects significantly elevates CSF levels of L-DOPA, DA and the DA metabolite dihydroxyphenylacetic acid (DOPAC) from age-matched control subjects (Figure 2A, B and C). There is a positive correlation of CSF DA levels to CSF L-DOPA levels. CSF NE levels in PD subjects on chronic L- DOPA treatment are not statistically different from age-matched controls (Figure 2B), but the NE metabolite 3,4-dihydroxyphenylglycol (DHPG) is significantly elevated in PD patients on chronic L-DOPA (Figure 2C). It is unclear why NE levels do not correlate to CSF L-DOPA levels in PD subjects like DA. A possible reason for the discrepancy in NE and DA levels in PD subjects on chronic L-DOPA therapy may be because administered L-DOPA is decarboxylated to DA in striatal neurons (as compared to DA from SN terminals) and serotonergic neurons, not in the few remaining dopaminergic neurons (Huot & Parent, 2007; Yamada et al., 2007). Orthostatic hypotension, which is attributed to reduced CNS NE function, is relieved only with the administration of DOPS (Goldstein et al., 2011). DOPS is also used to normalize CNS levels of NE in the DBH knockout mouse that lacks the synthetic enzyme DBH that converts DA to NE (Figure 1) (Thomas et al., 1998). So these data would suggest that NE function in untreated PD and possibly treated subjects is reduced.

Fig. 2. CSF catecholamine levels in PD subjects on chronic L-DOPA treatment and agematched control subjects. CSF spinal fluid was taken at the same time of day, approximately 4 hours after administration of L-DOPA. A) L-DOPA level, B) DA and NE levels and C) DOPAC and DHPG CSF levels in control and PD subjects on L-DOPA treatment. Catecholamine levels were extracted by alumina extraction and analyzed by high performance liquid chromatography (HPLC) as previously described (Szot et al., 2010). Each point represents a single individual catecholamine value for control and PD subjects, with average and standard error means (SEM) illustrated in bars for each catecholamine analyzed. \* Indicates significant difference between control and PD subjects.
