The number of mitochondrial proteins in mammals has been estimated to be
about 1300. To date, pathogenic defects have been found in only a fraction of
these proteins. Primary mitochondrial disease refers to disorders whose
underlying genetic cause directly impairs RC composition or function. Secondary
OXPHOS dysfunction, by contrast, has been described in a host of other genetic
or environmental disorders, including other genetic disorders (i.e., Rett
syndrome, other metabolic defects, chromosomal aneuploidies) or toxicities from
drugs (i.e., valproate, statins, pesticides).
The minimal prevalence of primary mitochondrial disease is one in 5000,
although pathogenic mutations in mitochondrial DNA (mtDNA) may occur as
frequently as one in 200 births. In the near future, developments in next
generation sequencing technologies will make it possible to include high
throughput sequence analysis in the diagnostic work-ups of mitochondrial
patients. Often, the prediction of the pathogenicity of unknown genetic variants is
not possible on the basis of sequence information alone. Usually, the level of
heteroplasmy of the mtDNA variant is checked in different tissues of the patient
and, in addition, in family members in the maternal lineage.
Establishing a diagnosis in patients with a suspected mitochondrial disorder is
often a challenge. Both knowledge of the clinical spectrum of mitochondrial
disorders and the number of identified disease-causing molecular genetic defects
are continuously expanding. The diagnostic examination of patients requires a
multidisciplinary clinical and laboratory evaluation in which the biochemical
examination of the mitochondrial functional state often plays a central role. In
most cases, a muscle biopsy provides the best opportunity to examine
mitochondrial function. In addition to activity measurements of individual
oxidative phosphorylation enzymes, analysis of mitochondrial respiration,
substrate oxidation, and ATP production rates is performed to obtain a detailed
picture of the mitochondrial energy-generating system. On the basis of the
compilation of clinical, biochemical, and other laboratory test results, candidate
genes are selected for molecular genetic testing. In patients in whom an
unknown genetic variant is identified, a compatible biochemical phenotype is
often required to firmly establish the diagnosis.
Metabolite analysis
Prior to the biochemical examination of a muscle biopsy, metabolite analysis in
blood and urine is usually performed. The results often provide important clues
for the presence of a mitochondrial defect and, in some cases, can even give
some indications for the location of the primary cause of the disease. Defects in
the mitochondrial energy-generating system may lead to high lactate levels in
blood, urine, and/ or CSF due to reduced pyruvate utilization by the mitochondria.
In the case of a respiratory chain defect, the lactate/pyruvate ratio in blood will
increase because of a shift in the mitochondrial redox state.
Enzyme measurements
Biochemical diagnostic examination of tissue and cell samples from
mitochondrial patients includes measurements of enzyme activities of the
oxidative phosphorylation (OXPHOS) system, consisting of complex I (EC
1.6.5.3), complex II (EC 1.3.5.1), complex III (EC 1.10.2.2), complex
IV (EC 1.9.3.1), and complex V (EC 3.6.1.3) (Fig. 1). Assays to quantify
OXPHOS enzyme activities are usually based on spectrophotometry.
Parkinson’s disease (PD),
the most common movement disorder, is characterized by age-dependent
degeneration of dopaminergic neurons in the substantia nigra of the mid-brain.
Non-motor symptoms of PD, however, precede the motor features caused by
dysfunction of the dopaminergic system, suggesting that PD is a systemic
disorder.
Mitochondrial dysfunction has long been observed in PD patients and animal
models, but the mechanistic link between mitochondrial dysfunction and PD
pathogenesis is not well understood. Recent studies have revealed that genes
associated with autosomal recessive forms of PD such as PINK1 and Parkin are
directly involved in regulating mitochondrial morphology and maintenance,
abnormality of which is also observed in the more common, sporadic forms of
PD, although the autosomal recessive PDs lack Lewy-body pathology that is
characteristic of sporadic PD. These latest findings suggest that at least some
forms of PD can be characterized as a mitochondrial disorder. Whether
mitochondrial dysfunction represents a unifying pathogenic mechanism of all PD
cases remains a major unresolved question.
Impairment of the ubiquitin–proteasome pathway can induce the accumulation of
reactive oxygen species in mitochondria, with the affected mitochondria later
removed by the autophagy pathway. In addition to impaired mitophagy,
decreased mitochondrial biogenesis, which may be closely linked to the TORmediated protein translation pathway, is also implicated in PD pathogenesis.
Thus, pathways for protein synthesis, quality control, mitochondrial maintenance,
and mitochondrial dynamics are mechanistically inter-connected in the
pathogenesis of PD, and represent novel targets for disease
prevention and treatment.
The loss of autophagy-related genes results in neurodegeneration and abnormal
protein accumulation. Autophagy is a bulk lysosomal degradation pathway
essential for the turnover of long-lived, misfolded or aggregated proteins, as well
as damaged or excess organelles. The accumulation and aggregation of synuclein is a characteristic feature of PD. Over-expression of -synuclein is
thought to impair autophagy, suggesting the presence of a cycle of impairment
and accumulation. Prior studies have shown that -synuclein is degraded by
chaperone-mediated autophagy.
Lewy bodies observed in PD brain tissue are proteinaceous intracellular
inclusions containing ubiquitin and -synuclein among many other components.
The protein Parkin, mutated in the most common cause of recessive PD, may
mediate the clearance of abnormal mitochondria through autophagy. Recent
studies have revealed that genes associated with autosomal recessive forms of
PD such as PINK1 and Parkin are directly involved in regulating mitochondrial
morphology and maintenance, abnormality of which is also observed in the more
common, idiopathic forms of PD. Note however that the autosomal recessive
PDs lack Lewy-body pathology that is characteristic of idiopathic PD.
Neurotoxins affecting humans and also used in animal models of PD:
MPTP, 6-hydroxy-dopamine (6-OHDA), rotenone, and paraquat
MPTP, a selective inhibitor of PD mitochondrial complex I, directed researchers’
attention to pathological roles of mitochondria in PD and raised the possibility
that environmental toxins affecting mitochondria might cause PD. Other
mitochondrial toxins characterized as parkinsonism-inducing reagents include 6OHDA, rotenone, and paraquat. Studies of animal models of PD induced with
these toxins suggest that mitochondrial dysfunction and oxidative stress are
important pathogenic mechanisms. In humans, reduced complex I activity has
been reported in both post-mortem brain samples and platelets of
sporadic/idiopathic PD cases.
Mitochondrial Cocktails (see M.J. Falk, 2010, (J Dev Behav Pediatr 31:610 –621)
Empiric “mitochondrial cocktails” are often prescribed at great expense to
families but without ability to objectively monitor clinical response or adverse
effects. No standard cocktail is universally used, although several vitamin and
antioxidant components are commonly included.
Common vitamin supplements include thiamine (B1), riboflavin (B2), ascorbate
(C), or B complexes (B50 or B100). Antioxidant supplements may include a wide
range of CoQ10 formulations and doses, lipoic acid, and tocopherol (vitamin E).
Although CoQ10 has clear benefit for the small subset of individuals with primary
Coenzyme Q deficiency, little evidence exists to suggest global benefit in all
mitochondrial RC diseases. Indeed, CoQ10 has both pro- and antioxidant
properties. Intermediary metabolic modifiers inconsistently recommended for RC
disease patients include L-creatine for individuals with myopathy, L-carnitine for
individuals with carnitine deficiency or some mitochondrial DNA cytopathies, and
folinic acid for individuals with secondary folate deficiency.
L-Arginine is worthy of special mention for its apparent utility in patients with
mitochondrial encephalopathy, lactic acidosis, and stroke to mitigate the
neurologic sequelae of metabolic stroke if administered intravenously within 30
minutes (and perhaps up to 24 hr) of acute neurologic manifestations, as well as
to reduce stroke frequency and severity with chronic oral administration.
Additionally, secondary CSF folate deficiency in mitochondrial disease may
present with a progressive leukoencephalopathy and white matter T2
hyperintensities evident on brain and spinal MRI; this finding can be highly
responsive to folinic acid, which crosses the blood–brain barrier to replenish CSF
folate.
The ketogenic diet is a matter of active controversy for the treatment of
intractable epilepsy in RC (respiratory chain) disease, because although it
provides an alternative fuel source (ketones) to bypass glycolysis, increases
Complex II-dependent respiration, and has been shown to delay progression in a
mouse model of mitochondrial myopathy, it has been associated with low
adherence, occasional lethality, and increased morbidity and mortality in an
MTERF2 mouse model having impaired mtDNA transcription. Therapeutic
monitoring of many “mitochondrial cocktails” is largely relegated to subjective
observations of clinical benefit and tolerance, commonly with chronic use of initial
doses despite obvious symptomatic progression. A recent review of
mitochondrial cocktail components’ indications, contraindications, adverse
effects, and dosing regimens for pediatric and adult patients with mitochondrial
disease has been published by the Mitochondrial Medicine Society. Emerging
therapeutic agents for which there is promising research in animal models and/or
early-stage clinical trials in humans include mitochondrial-targeted drugs to
increase their bioavailability at the site at which they are needed, lipophilic
antioxidants such as probucol, transcriptional modulators, and gene-therapy
aimed at mitochondrial delivery of restriction endonucleases to selectively
degrade mutant mtDNA and allow cell repopulation with normal mtDNA.