Investigating complex I deficiency in Purkinje cells and synapses in patients with mitochondrial disease

Abstract

Aims: Cerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus.

Methods: We investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age-matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses.

Results: Our data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses.

Conclusions: This is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia.

Keywords: Purkinje cells; ataxia; mitochondrial DNA; mitochondrial disease; respiratory chain deficiency; synapses.

Figure 1

Detecting and quantifying mitochondrial protein expression in Purkinje cells. Quadruple immunofluorescence of cerebellar Purkinje cells against Glutamic acid decarboxylase (GAD‐65/67), synaptophysin (SY‐38), complex IV subunit 4 (COX4) and complex 1 alpha subcomplex subunit 13 (NDUFA13). GAD‐65/67 is used to detect GABAergic cell bodies and inhibitory synapses. Combined with SY‐ 38 indicates the convergence of inhibitory presynaptic terminals. COX4 is a mitochondrial mass marker, and NDUFA13 protein is used to detect complex I deficiency. Control Purkinje cells demonstrate co‐localization of COX4 and NDUFA13 protein, indicating intact complex I expression, while NDUFA13 protein is significantly reduced in patient Purkinje cells (complex I‐deficient). Purkinje cells in the three examples shown above (Patients 1, 8 and 11) exhibit variably decreased NDUFA13 protein relative to the amount of COX4 present. Scale bar: 7 μm.

Figure 2

Detecting synaptic mitochondria and reconstructing GABAergic synapses. Development of the protocol for detecting and reconstructing GABAergic presynaptic terminals onto dentate nucleus neurons. Top panel: Confocal images of immunofluorescently stained control dentate nucleus neurons were used to identify inhibitory synapses. Scale bar: 7 μm. Magnified view: Areas around the periphery of neurons where GAD‐65/67 (blue) and synaptophysin (purple) puncta coexist are denotative for the presence of inhibitory synapses. COX4‐positive staining (red) indicates mitochondrial mass, and NDUFA13 presence (green) is used to detect respiratory chain protein deficiency. Scale bar: 1.5 μm. Object detection: Actual staining substituted by objects that represent each of the four markers used. Mitochondrial objects can then be compartmentalized and filtered so that only the ones that are within synapses are studied (far right). Bottom panel: Three‐dimensional reconstruction of inhibitory synapses on the same control neuron, and identification of synaptic mitochondria. Scale: 1 unit = 1.56 μm. Protocol development is achieved using the Volocity® software (v.6.3.1, PerkinElmer).

Figure 3

NDUFA13 protein expression in Purkinje cells and their inhibitory synapses on the dentate nucleus. Complex I deficiency in patient cerebellar Purkinje cells and inhibitory synapses is expressed as z score values. Coloured box plots represent Purkinje cell z values, and clear boxplots reflect inhibitory synapse z values. In Purkinje cells, the most severe deficiency is generally observed in patients with the m.3243A > G point mutation (Patients 1–6), with Patient 5 (m.3243A > G) showing the most dramatic loss of NDUFA13 protein expression (Median z score = −12). Two individuals with recessive POLG mutations (Patient 9 and 10) demonstrate relatively high NDUFA13 protein expression. Patients with the same genetic defect show variability in their complex I expression (Patient 2 vs. Patients 1 and 3 for m.3243A > G; Patient 7 vs. Patient 8 for m.8344A > G). In inhibitory synapses, the majority of patients revealed variably decreased complex I expression, with Patients 5 (m.3243A > G) and 11 (POLG) possessing the most deficient synaptic terminals, while Patients 2 (m.3243A > G) and 10 (POLG) are not different from controls.

Figure 4

Complex I deficiency in inhibitory synapses on dentate nucleus neurons. Quantification of NDUFA13 protein expression in GABAergic synapses around dentate nucleus neurons (top panel; scale bar: 7 μm). Control inhibitory synapses have equal expression of COX4 (mitochondrial mass) and NDUFA13 (white arrows – left panel), indicating intact complex I expression. In contrast, mitochondria in patient inhibitory presynaptic terminals have decreased complex I expression (white arrows), indicating mitochondrial dysfunction in these synapses. There is a severe reduction in complex I expression relative to mitochondria (Patient 5; m.3243A > G), while in other patients, there is only a small difference in complex I to COX4 expression (Patient 11; POLG). Scale bar: 1.5 μm.

Figure 5

Correlation between NMDAS score for cerebellar ataxia and NDUFA13 protein expression in inhibitory presynaptic terminals. More severe ataxia scores are related to lower z scores for NDUFA13 expression (Spearman's rho = −0.60, P value = 0.0493).

Figure 6

Changes in the number of inhibitory presynaptic terminals. The number of GABAergic synapses counted on each dentate nucleus neuron (n = 20) expressed as z score values. Decreased numbers of inhibitory presynaptic terminals is detected in eight out of 12 patients. All patients with recessive POLG mutations exhibit synapse loss, in addition to the majority of patients harbouring the m.3243A > G point mutation. Intriguingly, Patients 3 (m.3243A > G), 8 (m.8344A > G) and 12 (m.14709T > C) have increased numbers per cell compared with controls.

Figure 7

MtDNA mutation load in isolated Purkinje cells and inhibitory presynaptic terminals. Heteroplasmy levels for individually isolated Purkinje cell bodies and inhibitory presynaptic terminals from five patients harbouring mtDNA point mutations (m.3243A > G and m.8344A > G). Individual symbols represent independent read‐outs, and bars represent medians (n = 15 for both populations).